Rabbit monoclonal anti gapdh hrp conjugate

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.