Anti tubulin mouse monoclonal antibody

hPDLSCs-OE-GRP78 were grown under normal growth and differentiation conditions with or without DMP1 treatment for 24 h. Cells were then harvested and lysed in RIPA buffer (cell signalling) containing protease and phosphate inhibitor cocktail (Millipore). Centrifugation was then performed at 11,500 ×g for 15 min at 4° C and the supernatants were used as total cellular proteins. Protein concentrations were measured using the Bio-Rad Protein Assay Dye Reagent (BIO-RAD) with BSA as standard. 25 μg of total proteins were loaded on a 10 % SDS-polyacrylamide gel. The proteins were transferred onto a nitrocellulose membrane following electrophoresis, blocked with 5 % skim milk (Merkel et al., 2019 (link)). The membranes were incubated with the following primary antibodies: anti-TRIP-1 rabbit polyclonal antibody (1/1000; Invitrogen), anti-FL DMP1 (1/1000; house-made), anti-DPP rabbit polyclonal antibody (1/1000; house-made) (Eapen et al., 2012 (link)). Anti-tubulin mouse monoclonal antibody (1/5000; Invitrogen) was used as a loading control. The blots were incubated in either anti-mouse or anti-rabbit secondary conjugated with HRP. Each of the blots were washed 4 times with PBS, and the bands were visualised using chemiluminescence detection (Thermo Fisher Scientific) using X-ray films according to the manufacturer’s protocol.