Rabbit polyclonal anti α tubulin

Western blotting was performed as previously described [17 (link)]. In brief, protein concentrations of samples were determined using the BCA protein assay (Bio-Rad, Hemel Hempstead, Herts, UK). Twenty micrograms of each sample were subjected to Western blot analysis using the following primary antibodies: rabbit polyclonal anti-phospho-tyrosine receptor kinase B (TrkB) (phospho Y515, abeam, Cambridge, MA, USA) at 1:1000 dilution, rabbit polyclonal anti-TrkB (abeam) at 1:1000 dilution and rabbit polyclonal anti-α-tubulin (cell signaling Technology Inc.) at 1:1000 dilution. Images were scanned by an Image Master II scanner (GE Healthcare, Milwaukee, WI, USA) and analyzed using ImageQuant TL software v2003.03 (GE Healthcare). The band signals of the interesting proteins were normalized to those of the corresponding α-tubulin and expressed as fractions of control sample from the same gels.

For western blot analysis, cells or tissues were lysed in RIPA buffer (50mM TRIS pH:7.50, 25mM NaF, 100mM NaCl, 5mM EDTA, 0.1% SDS, 1% TritonX-100) supplemented with protease and phosphatase inhibitors, 20mM nicotinamide and 20μM vorinostat. Equal amounts of total-lysates were separated on 4–15% SDS-PAGE gels (Bio-Rad), transferred to PVDF membranes (Millipore), and probed with indicated antibodies. Blots were washed with PBS/T (0.1% Tween-20 in PBS) and either developed (for GAPDH-HRP) or probed with HRP-conjugated secondary antibodies (Cell Signaling, Cat# 7074 and 7076), and developed. Rabbit monoclonal anti-HDAC6 (Cat# 7612), Rabbit monoclonal anti-Acetyl-α-Tubulin (Cat# 5335), Rabbit polyclonal anti-α-Tubulin (Cat# 2144), Rabbit monoclonal anti-GAPDH HRP conjugate (Cat# 8884) were from Cell Signaling (Danvers, MA). Rabbit monoclonal anti-HDAC6 (Cat# 7612), Rabbit monoclonal anti-Acetyl-α-Tubulin (Cat# 5335) antibodies were validated using wild-type and HDAC6 KO mouse tissues as in Extended Data Fig. 2b, c. Primary antibodies were used at 1:1000 dilution with anti-Acetyl-α-Tubulin antibody used at 1:10,000. Secondary HRP-conjugated antibodies were used at 1:10,000 dilution. Western blot images were quantified using Adobe Photoshop SC6 and ImageJ version 1.51.