Anti gapdh 6c5 mab

Immunoblotting was performed as previously described (15 (link),16 (link)). In brief, cells were lysed with RIPA lysis buffer supplemented with a protease and phosphatase inhibitor cocktail (both from Nacalai Tesque, Kyoto, Japan). Cellular proteins were quantified using a DC Protein Assay kit (Bio-Rad, Hercules, CA, USA). Equal amounts of proteins were loaded onto the gels, separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and transferred onto Immobilon-P membrane (Millipore, Billerica, MA, USA). The membranes were probed with primary antibodies (Abs) such as anti-acetylated-α-tubulin (6–11B-1) mAb, anti-α-tubulin (B-7) mAb, anti-GAPDH (6C5) mAb, anti-HDAC6 (H-300) Ab, and anti-Ub (P4D1) mAb, which were all purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA) and anti-PARP Ab (Cell Signaling Technology, Inc., Danvers, MA, USA). Immunoreactive proteins were detected with horseradish peroxidase-conjugated secondary Abs (Cell Signaling Technology, Inc.) and an enhanced chemiluminescence reagent (Millipore). Densitometry was performed using a Molecular Imager ChemiDoc XRS System (Bio-Rad).