Mouse anti α tubulin monoclonal antibody

Cells were harvested, washed and lysed by lysis buffer. Proteins were separated by SDS/PAGE (8% gel) and transferred to nitrocellulose membrane. After blocking in 5% non-fat milk for 1 h, the membranes were incubated with the following primary antibodies:rabbit anti-NRP1 monoclonal antibody (1:1000, Cell Signaling Technology), mouse anti-α tubulin monoclonal antibody (1:1000, Abcam). The proteins were visualized with enhanced chemiluminescence reagents (Pierce).

P3HR1 cells were lysed in mRIPA buffer (50 mM Tris-HCl, pH 7.8, 150 mM NaCl, 5 mM EDTA, 0.5% Triton X-100, 0.5% NP-40) [26 (link),27 (link)]. Approximately 5% of the lysate was loaded to a gel and then separated by electrophoresis in 10% SDS-PAGE. The proteins in the gel were electroblotted onto a polyvinylidene difluoride (PVDF) blotting membrane while using a Hoefer transfer system. Rta was detected while using mouse anti-Rta monoclonal antibody (Argene, Verniolle, France). α-Tubulin was detected using mouse anti-α-tubulin monoclonal antibody (Abcam, Cambridge, UK). Primary antibodies were detected using horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology, Danvers, MA, USA) and then visualized using SuperSignal West Pico Chemiluminescent substrate (Thermo Fisher Scientific, Waltham, MA, USA).