More specialised variants of ELS are available for organic farming and severely disadvantaged areas in the uplands. Although management measures in ELS have been demonstrated to benefit pollinators, such as nectar flower mixes and low input pastureland (Scheper et al. 2013), payments for ELS are fixed regardless of the combination of CP673451 order options used to qualify and therefore uptake has typically been biased towards lower PF-02341066 solubility dmso cost, often opportunistic options (e.g. low frequency hedge cutting), that are thought to be less beneficial to biodiversity (Sutherland 2009; Hodge and Reader 2010). Furthermore, much of this uptake has been in low

productivity areas where AES are thought to be less beneficial due to high existing habitat diversity (Hodge and Reader 2010; Scheper et al. 2013; Cloither 2013). Like all AES, the monitoring of ELS is limited by its budget, allowing for potentially high levels of poor or false implementation (Kleijn and Sutherland 2003) and can vary strongly in their effectiveness

between scheme designs (Kleijn et al. 2006). Recently accepted reforms to CAP include a greening requirement in order to claim the full value of subsidies. This includes a mandatory 5 % of land to be designated as ecological focus areas, comprised of a combination of hedges, trees, fallow land, grassland maintenance and low input margins (European Commission 2013). Although this may result in ELS being replaced or radically overhauled, there is still a need to appraise benefits of the current management options under the scheme in order to better inform potential successors. Whilst evidence exists selleck screening library to suggest ELS options can improve the quality of insect pollinator habitats (Kleijn et al. 2006; Potts et al. 2009; Pywell et al. 2011), the

benefits of most options remain unknown, and are likely to remain so given the significant Dimethyl sulfoxide investment and time in conducting robust empirical studies. Furthermore, although economic valuations of pollination services have been used to justify expenditure on mitigation efforts, to date only one study has compared these benefits to any costs of conservation actions (Cook et al. 2007). The purpose of this study is therefore twofold; first, to provide a simple appraisal of the relative benefits of all ELS options to providing good quality pollinator habitat. Secondly this study provides an estimate of the cost in adapting the currently utilised ELS area towards pollinator conservation provision by redistributing the current national mix of ELS options towards one reflective of the relative benefits to insect pollinator habitat. Methods This study focuses upon the entry level stewardship (ELS) as it is both very widespread, incorporating 5 M ha of English Farmland (Natural England 2013a), and has many options that are applicable to other UK and European agri-environment schemes (AES).

The positive association between maternal age and risk of fractures is difficult to interpret. Our original hypothesis was that children of adolescent mothers

might have been at greater risk due to inadequate child care, but the results came out in the opposite direction. It is possible that older mothers have faced increased demands on calcium and vitamin D stores through repeated pregnancies, which could explain the positive association between maternal age and risk of fractures. However, adjustment for parity did not influence such an association. We found no other studies reporting such an association and confirmation by other researchers is essential. A previous study in the same city reported that adults in

the lowest socioeconomic position Apoptosis inhibitor category—based on household assets—were 3.2 times more likely than those in the highest category to have experienced a fracture within the 12 months prior to the interview [17]. Because the socioeconomic classification is based on assets acquired over several years rather than concurrent income, reverse causality is unlikely to explain this finding. Data from the ALSPAC cohort in the United Kingdom showed that social position is directly related to bone mineral content of adolescents [18], which may reduce Eltanexor purchase their risk of fractures. These trends were not confirmed in our study with Brazilian adolescents. In the Poisson models, the association was actually in the opposite direction. A limitation of our study is that, so far, we have no data on bone mineral density for cohort members. We are planning to collect such data in the next follow-up visit, which will take place in 2011, when subjects will be aged 18 years. An advantage of our study is that two multivariable techniques provided consistent results in terms of the risk factors for fractures, reducing the possibility of type 1 error. Also, the prospective nature of the data reduces the possibility of recall

bias. Our findings are in agreement with the literature regarding an increased risk of fractures among boys and among children who were longer at birth [8, 18, 19]. The finding on higher risk among children born to older mothers needs to be Amino acid replicated. Our results suggest that, in accordance with the hypothesis of developmental origins of diseases, fractures seem to be, at least in part, programmed in early life. Acknowledgements This analysis was supported by the Wellcome Trust initiative entitled Major Awards for Latin America on Health Consequences of Population Change. Earlier phases of the 1993 cohort study were funded by the European Union, the 3-MA order National Program for Centers of Excellence (Brazil), the National Research Council (Brazil) and the Ministry of Health (Brazil). Conflicts of interest None.

For men, five of the seven plasma indices were significantly associated with hand grip strength, but for women, none of the seven were associated with this index of physical function. For men, the pattern of associations with a physical activity score was similar to that for grip strength, and for women, four of the plasma indices were associated with the physical activity score. For men, three of the plasma indices were associated Adavosertib molecular weight with smoking habit,

but for women, only one (plasma phosphorus) was associated with this lifestyle index. Plasma PTH was not significantly correlated with any of the function and lifestyle indices (not shown). Table 2 Linear

regression of plasma bone-related indices versus selected functional and lifestyle indices   Versus hand grip strengtha,b Versus physical activity scorea,c Versus smoking habita,d t value P t value P t value P Plasma indices: see more men  P-calcium −0.4 0.7 +1.2 0.2 −1.1 0.3  P-phosphorus −2.2 0.03 +2.4 0.015 −0.2 0.9  P-25(OH)D +3.5 0.0005 −3.3 0.001 −2.4 0.02  P-alkaline phosphatase −2.1 0.04 +1.4 0.15 +3.7 0.0003  P-albumin +2.7 0.007 −1.9 0.05 −1.1 0.3  P-creatinine −0.7 0.5 +2.0 0.04 +0.3 0.7  P-α1PF-02341066 chemical structure -antichymotrypsin −2.8 0.005 +3.0 0.003 +4.4 <0.0001 Plasma indices: women  P-calcium −0.8 0.5 −0.8 0.4 +0.5 0.6  P-phosphorus −0.9 0.4

−0.8 0.4 +2.5 0.01  P-25(OH)D +1.6 0.12 −4.1 <0.0001 −1.8 0.08  P-alkaline phosphatase −0.4 0.7 +2.3 0.02 +1.3 0.2  P-albumin +0.2 0.8 −4.2 <0.0001 +0.9 0.4  P-creatinine −0.5 0.6 −0.3 0.7 +0.1 0.9  P-α1-antichymotrypsin −1.5 0.12 +2.3 0.02 +1.6 0.1 aRegressions adjusted for age and confined to those subjects for whom mortality data were available. Alkaline phosphatase, creatinine and α1-antichymotrypsin were log-transformed before the analyses. df = 378–435. P plasma bContinuous variable: mean estimate Metalloexopeptidase for both hands. Higher values denote greater hand grip strength [5] cFour discrete categories: from 1 = very active to 4 = very inactive [5] dThree discrete categories: 0 = non-smoker; 1 = <20 cigarettes/day; 3 = >20 cigarettes/day [5] Hazard ratios for all-cause mortality Table 3 lists the age- and sex-adjusted hazard ratios for all-cause mortality for both sexes combined and subdivided by sex. For the combined sexes, significant predictors of mortality included plasma 25(OH)D (‘protective’), plasma phosphorus (‘deleterious’, i.e. higher levels = greater risk) and dietary energy (‘protective’).

These techniques include thermal evaporation [5, 29], hydrothermal [2, 3] and electrochemical deposition [4], and metal-organic vapor-phase epitaxy (MOVPE) [1]. In this paper, we report the seed/catalyst-free growth of ZnO structures on multilayer (ML) graphene by thermal evaporation. The dependence of substrate temperatures on the properties

of grown structures was studied. Based on the obtained results, a growth mechanism was proposed. Methods A ML graphene on SiO2/Si (Graphene Laboratories Inc, Calverton, NY, USA) was learn more used as a substrate. Figure  1a shows the measured Raman spectra of the ML graphene. The 2D peaks at approximately 2,700 cm-1 of the Raman spectra for graphite as shown by locations 1 and 4 have broader and up-shifted 2D band indicating few layer graphene [30]. Figure  1b shows the schematic of the experimental setup. The growth was carried out by thermal evaporation PF-01367338 research buy technique in dual zone furnace. High-purity metallic Zn powder (99.85%) and oxygen (O2) gas (99.80%) were used as the sources. Prior to the growth process, the substrate was treated with organic cleaning of ethanol, acetone, and deionized (DI) water to remove any unwanted impurities on the substrate. Zn powder of approximately 0.6 g was spread evenly into a ceramic boat. The ceramic boat was placed in the zone 1 of the furnace, while the substrate was placed inclined at 45°

in the zone 2 of the furnace. The distance between source and substrate was fixed at 23 cm. Two independent temperatures were applied to the Selleck NCT-501 furnace system. Here, T1 denotes to the set temperature (ST) of the source while T2 denotes to the ST of the substrate. Firstly, the temperature of zone Clomifene 2 was raised to T2 (i.e., 600°C, 800°C, or 1,000°C) in argon (Ar) environment (Ar flow rate of 200 sccm).

Then, the temperature of zone 1 was raised to T1 (1,000°C). The flow of Ar was stopped when the temperature of zone 1 reached 400°C (Zn melting point, 419°C). This was done in order to avoid the transfer of Zn particles to substrate prior to actual growth. The heating of Zn powder was continued until it reached 1,000°C. It was confirmed from several attempts that such high temperature was needed for continuous and constant evaporation of Zn. After reaching 1,000°C, O2 (400 sccm) was introduced for 1 h of growth time. Finally, the furnace was turned off and the samples were cooled down to room temperature. Figure  1c summarizes the growth procedures. The as-grown ZnO was examined using field-emission scanning electron (FESEM) microscopy (SU8030, Hitachi, Chiyoda, Tokyo, Japan), dispersive X-ray (EDX) spectroscopy, X-ray diffraction (XRD) (Bruker, AXES, D8 Advance, Bruker Corporation, Billerica, MA, USA) and photoluminescence (PL) spectroscopy (Horiba JobinYvon, Tokyo, Japan). Figure 1 Raman spectra of ML graphene (a), schematic of growth setup (b), and growth time chart (c).

In addition, an A-T rich region is found upstream of this sequence, strongly suggesting a role for these sequences in the binding of the IHF protein. Mobility shift assays with mutant probes clearly demonstrated a role for these residues in the P phtD -IHF interaction. Similarly, our proposal for the requirement of a change in the DNA structure Proteasome inhibitor for IHF binding to the phtD operon is somewhat supported

by various reports which demonstrate that besides the interaction with consensus sequences, the IHF protein requires a curved DNA structure for binding [38]. The IHF protein contributes in an important way to the function of a wide variety of macromolecular processes 3-MA in vitro in bacteria and is recognized as a global regulation factor in the transcription of many genes. IHF can alter gene expression in a number of ways, including positive and negative effects on transcription, and its role as a regulator of virulence gene expression has increasingly been determined [39, 42]. The role of IHF protein in regulating phtD operon expression was examined through the analysis of a phtD::gfp

transcriptional fusion in an E. coli K12 ihfA – mutant background, which clearly showed higher transcriptional activity than that observed in the wild type background. This activity significantly decreases when the ihfA – mutant strain is complemented in trans with the ihfA gene of P. syringae pv. phaseolicola NPS3121, suggesting that the IHF protein has a negative effect on the expression of the phtD operon in E. coli. Because some reports have demonstrated that the E. coli IHF protein can functionally replace IHF proteins of some Pseudomonas Amino acid species, and since this protein is not modulated by interactions with inducer or co-repressor molecules, as are most transcription factors [33, 35], we propose that the IHF protein also exerts a negative effect on P. syringae pv. phaseolicola

NPS3121 phtD operon expression. IHF has been shown to act as a negative regulator through several mechanisms. In some cases, IHF seems to act as a classical repressor by binding to DNA within the RNA polymerase recognition site and excluding the polymerase from the promoter. IHF may also act indirectly as a repressor, ABT-737 datasheet collaborating with a gene-specific repressor or obstructing the binding of an activator. Alternatively, IHF can repress transcription in concert with other nucleoid proteins and global or gene-specific transcriptional regulators to create a higher-order nucleoprotein complex that forms an inhibitory promoter architecture [35, 37, 42]. The way in which IHF could act to repress the phtD operon is unknown, although according to the position of the predicted IHF binding site (-64 to -44), its role as a classical repressor may be dismissed.

Prolonged exercise at high intensities leads to a quantitative redistribution of blood flow to the exercising muscle (exercise hyperthermia) in proportion to its energy demands of oxygen and

substrates. Sympathoadrenal activity, however, see more reduces water and sodium loss during exercise by decreasing renal blood flow and changing its distribution by direct tubular effects. Moreover, it decreases Foretinib potassium loss by facilitating its muscular uptake [22]. Blood flow to the skin is increased to facilitate heat dissipation, and sweating implies loss of water and electrolytes from the body. Dehydration of approximately 2-3% of body mass routinely occurs during intermittent high-intensity exercise, especially when the ambient temperature is high. Usually, thirst is triggered when the individual is already 5% dehydrated [23]. The dehydrated state can buy Selumetinib be worsened by catecholamine-induced thirst suppression [24]. Fluid loss results in decreasing circulatory blood volume, blood pressure,

sweat production and stroke volume, as result, vascular resistance increase leading to a skin blood flow decreased, all of which impair heat dissipation. Heart rate rises to some additional 3-5 beats/minute for every 1% body weight loss due to dehydration [25]. Dehydration has a negative effect on endurance performance by increasing muscular glycogen degradation and plasma lactate levels and by causing cardiovascular drift and reduced ability to transport heat to the periphery for dissipation, thus resulting in increased core temperature

[26]. 3.1 Exercise-dependent, dehydration-induced hyperthermia Heat production during exercise is 15-20 times greater than at rest, and it is sufficient to raise core body temperature by 1°C every 5 minutes if there are no thermoregulatory adjustments [25]. The body’s multiple mechanisms for heat dissipation to prevent significant hyperthermia include conduction, convection, evaporation and radiation. As ambient temperature rises above 20°C, the contributions of conduction, convection this website and particularly radiation, become increasingly insignificant with the bulk of the heat dissipation during exercise resulting from evaporation as sweat. In hot, dry conditions, evaporation may account for as much as 98% of dissipated heat. Sweat evaporation leads to dehydration, which increases body temperature [25]. Any factor that limits evaporation, such as high humidity or dehydration will have profound effects on physiological function, athletic performance, and risk for heat illness [27]. There are five common types of heat illness, the milder forms including heat edema, heat cramps, heat syncope, and heat exhaustion. The most severe form of heat illness is heat stroke [28]. The milder forms of heat illness are widely underreported and underdiagnosed [25].

2 associated with high lactate concentration [27], Berzosertib in vitro whereas for SARA, where the condition is subtler, several definitions have been proposed [13, 28, 29]. For the purpose of this study, we used a mean value of 6.25 as the ruminal pH benchmark for SARA determination [30]. Based on the ruminal pH and fermentation patterns observed in this study during the 3-d feed challenge periods, acidosis induction was attained on d3 (data not shown). Lactic acidosis was induced with wheat, whereas butyric 10058-F4 clinical trial and propionic SARA were

induced with corn and beet pulp, respectively. These results are similar to those of our previous study [13] in which these three acidosis forms were induced in wethers using the same feeds. Irrespective of the acidosis, we also observed that the differences among treatments were accentuated during the three days of feed challenges, being maximal and significant only on the third day. Consequently, only data related to the effect of probiotic supplementations on the rumen characteristics on d3 are reported and discussed here. Lactic acidosis induced by wheat Lactic acidosis is a rare accidental pathology in which the ruminal ecosystem is completely disturbed. In this experiment, the mean and minimum ruminal pH were 5.25 and 4.86 respectively, concentration of lactate reaching ~ 34 mM and that of total VFAs 94 mM for control wethers (Table 3). These values are classically observed in lactic acidosis situations [13, 31]. Compared

with the control animals, a drastic decrease in total bacteria was observed for Lr + P fed wethers (P < 0.05; Figure 1), whereas

feeding P and Lr + P decreased SIS3 in vitro the population of protozoa (P < 0.05). Without significantly affecting fibrolytic activities (cellulase and xylanase), the three probiotic treatments reduced the proportion of the cellulolytic bacterium F. succinogenes, Lr + P decreased R. albus while R. flavefaciens was not affected. The growth of lactate-producing bacteria (Lactobacillus spp. and S. bovis) was enhanced by probiotic supplementation. S. bovis Lenvatinib molecular weight proportion was highest for P-fed wethers whereas Lactobacillus spp. became a predominant bacterial group: from 1.7% in C up to 25% of total bacteria in probiotic-supplemented wethers (P < 0.05). Specific amylase activity was not significantly affected by probiotic supplementation, but the total activity was increased in P-fed wethers (P < 0.05; data not shown). As expected, lactobacilli proliferation caused an increase in lactate concentration that reached more than 60 mM in probiotic-fed wethers (P < 0.05; Table 3), whereas total VFA concentrations were less than 35 mM for P and Lr + P (P < 0.05), suggesting a decrease in microbial fermentative activity and a shift towards lactate production at the expense of VFAs (P < 0.05). It could be argued that the increase was due to the addition of exogenous lactobacilli. However, wethers that received only Propionibacterium P63 exhibited similar proportions of Lactobacillus spp.

We found that the nucleus import of PLAG1 was aided by KPNA2 and would amplify the transcriptional activities of PLAG1 in HCC. Several genes including IGF-II, CRABP2, CRLF1, CRIP2, which are transcriptional targets of PLAG1, could be up-regulated by enhanced KPNA2. IGF-II is frequently up-regulated in HCC and was enriched in the proliferation subclass of the molecular classification of HCC Sapitinib [27]. Besides, inhibition of IGF-II could impair the proliferation and invasive activities of HCC cells [20]. Furthermore, inhibition of PLAG1 in cell clones with stable KPNA2 over-expression

could abolish the up-regulation of these genes and could counteract the pro-tumoral effects of KPNA2. The result implied that downstream molecular of PLAG1 such as IGF-II might

be partly responsible for the role of KPNA2 in HCC. Although we revealed PLAG1 would be a critical mediator for KPNA2, it is noteworthy that whether other transcriptional factors carried into nucleus by KPNA2 might participate in HCC regulation need to be explored. Cancer classification using biomarkers may effectively define the risk of recurrence, which allows for the use of appropriate treatments to acquire a better prognosis. The prognosis of patients with positive KPNA2 expression could be clustered by the status of PLAG1 nucleus enrichment, validating that the biological effects of KPNA2 relied on the interaction with PLAG1. Besides, for the subgroup of patients with negative PLAG1 expression, selleck compound the prognostic value of

KPNA2 came to be lost, further confirming that inhibition of PLAG1 could significantly retard the role of KPNA2 in tumor growth and metastasis in vitro as shown in Figure 2b and 2d. Combined with nucleus enrichment of PLAG1, the positive KPNA2 status would be more accurate to predict the prognosis of HCC patients after hepatectomy. Patients with co-existence of positive KPNA2 expression check and positive PLAG1 expression should be closely monitored and receive appropriate adjuvant therapies. However, further investigation should be done to KU55933 cost validate the prognostic value of KPNA2 and PLAG1 in other cohort of HCC patients, which would be promising for clinical application to reduce the false positive rate to identify and monitor patients with high recurrent risk after hepatectomy. Conclusions PLAG1 could be impelled into nucleus by interaction with KPNA2, adapter acting in nucleus protein import. Co-enrichment of KPNA2 and PLAG1 in nucleus is observed in clinical samples. The increment of proliferative and metastatic abilities by KPNA2 can be significantly retarded by PLAG1 inhibition.

For bromeliads, the pattern was more variable, with highest species richness in the humid montane Yungas and dry inter-Andean forest, followed by Amazonian and Tucumano-Bolivian forest (Fig. 2). Epiphytic species were Selleck DAPT generally most common, and their frequency was highest in the dry vegetation types and relatively low in the Amazonian. In contrast to the aroids, useful bromeliads had a more restricted geographical distribution. While the proportion of widely and narrowly distributed species is more or less similar, the number of endemic species is significant (Fig. 3).

In general, bromeliads showed preferences for certain habitats in most of the ecoregions studied, although almost no preferences were found in the dry inter-Andean valleys (Fig. 4). Ornamental species were well represented

both in the humid montane and dry inter-Andean forests. Medicinal, multi-use, fiber-producing, and food species were most species-rich in the dry forests of the inter-Andean valleys such as the Gran Chaco and the Chiquitano forest (Fig. 5). Species used as food sources were also well represented in Amazonian forests. Discussion Bolivia has a striking number of potentially useful species of Araceae and Bromeliaceae, which can provide many non-timber forest products. 3-deazaneplanocin A Both families show distinct distribution patterns and ecological features indicating, thus, that their economic potential may differ among ecoregions. Araceae were most species-rich and most frequent in the humid

lowland and montane forest. This pattern is in accordance with their overall richness pattern (Valencia et al. 1994; Kessler and Croat 1999). Particularly, aroids with medicinal properties have a wide distribution and, for this reason, it is not surprising that this family is considered as one of the most EPZ5676 clinical trial commonly used liana and climbing plant families (Bennett 1992, 1995). Chorioepithelioma Some species, particularly those of Monstera, Syngonium, and Philodendron, which are most diverse in the lowlands, may be abundant in weedy situations (open habitats, road sides, fence rows, plantations) as a possible result of pre-adaptation to such conditions (Croat 1988). When comparing tropical lowland fallows with adjacent mature forests, species richness of aroids showed no reduction (Krömer and Gradstein 2003). Our study shows that the species of aroids most suitable for sustainable utilization are principally located in the Amazonian region. In other regions where species are less frequent, more specialised, and with a narrower distribution, their exploitation may be harmful for the natural populations and, hence, not feasible on a sustainable basis. Ecosystems with more diverse habitats, numerous plant species, and variable life forms, such as montane forests, are generally more vulnerable to human use (Wild and Mutebi 1996).

The extent of wound closure was examined by phase contrast microscopy with the LuciaG software (Laboratory Imaging s.r.o., Prague, Czech Republic) at time points 0, 3, 6, 9, 12 and 24 h. RNA isolation and quantitative real-time PCR Total RNA from cell culture was isolated by the Qiagen RNeasy Mini Kit (Qiagen, Hilden,

Germany) according to the manufacturer’s protocol. Synthesis of cDNA was performed using QuantiTect Reverse Transcription Kit (Qiagen, Hilden, Germany) with an extended incubation time of 30 min at 42°C. QRT-PCR was performed using an ABI 7500 Fast PCR instrument (Life Technologies, Darmstadt, Germany) with QuantiTect SYBR Green RT-PCR Kit (Qiagen, Hilden, Germany) according to the manufacture’s protocol. To determine the expression levels of HDAC1 (#QT00015239), HDAC2 (#QT00001890), HDAC3 (#QT00093730) and HDAC8 (#QT00049630) #17-AAG mw randurls[1|1|,|CHEM1|]# we used QuantiTect Primer assays (Qiagen, Hilden, Germany) at an annealing temperature of Epoxomicin supplier 55°C. The expression of the housekeeping gene TATA-box binding protein (TBP) was determined with self-designed primers (forward: 5’-ACAACAGCCTGCCACCTTA-3’; reverse: 5’-GAATAGGCTGTGGGTCAGT-3’). Technical duplicates had less than 10% standard deviation. Western blot analysis Western blot analysis of whole-cell extracts were done as described previously [39]. Total protein was

extracted by cell lysis in a RIPA-buffer containing 150 mM NaCl, 1% Triton X-100, 0.5% desoxycholate, 1% Nonidet P-40, 0.1% SDS, 1 mM EDTA, 50 mM Tris (pH 7,6) and a protease inhibitor cocktail (10 μl/ml, #P-8340, Sigma-Aldrich) for 30 minutes on ice. Protein concentrations were determined by BCA protein assay (Thermo Scientific, Rockford, IL). After separation in SDS-page gels and transfer to PVDF membranes (Merck Millipore, Berlin, Germany) the membranes

either were blocked with 5% non-fat milk in TBST (150 mM NaCl, 10 mM Tris, pH 7.4 and 0.1% Tween-20), washed and then incubated with primary antibodies at room temperature for 1 h or at 4°C over night. Primary antibodies were used against HDAC1 (1:1,000, C-19, sc-6298; Santa Cruz Biotechnology, Heidelberg, Germany), HDAC2 (1:5,000, H-54, sc-7899; Santa Cruz Biotechnology, Heidelberg, Germany), HDAC3 (1:1,000, H-99, sc-11417; Santa Cruz Biotechnology, Heidelberg, Germany), HDAC8 (1:400, A-4008; Epigentek, Brooklyn, NY), p21 (1:400, C-19, sc-397; Santa Cruz Biotechnology, Heidelberg, Germany), thymidylate synthase (1:1,000, TS, TS106, Millipore, Temecula, CA), PARP (poly [ADP-ribose] polymerase 1, 1:500, 46D11; Cell Signaling Technology, Inc., Danvers, MA) and acetylated α-tubulin (1:15,000, #T-7451, Sigma Aldrich, St. Louis, Mo). Anti-α-Tubulin B-512 (Sigma Aldrich, St. Louis, MO) was used as loading control in a concentration of 1:50,000.