Mouse monoclonal anti gapdh antibody

Protein extraction and Western blot were performed according to a previous study 29 with the following antibodies: a rabbit antibody against ZEB1 (1:1000, Abcam, Cambridge, MA, USA), the EMT antibody sampler kit (1:1000, Cell Signaling Technology, Danvers, MA, USA), the Src antibody sampler kit (1:1000, Cell Signaling Technology), a rabbit polyclonal anti‐CD133 antibody (1:1000, Proteintech, Rosemont, IL, USA), a mouse monoclonal anti‐GAPDH antibody (1:4000, Cwbiotech, Beijing, China) and a goat anti‐rabbit IgG conjugated with horseradish peroxidase (1:5000, Good‐Science, Shanghai, China). Visualization of protein bands was performed using the Tanon‐5200 system (Biotanon, Shanghai, China).

Whole cell lysates were extracted with PBS buffer supplemented with 1% Nonidet P-40, 1 mM EDTA, 1 mM PMSF, and 1× Protease Inhibitor Cocktail (Sigma). After SDS-polyacrylamide gel electrophoresis (SDS-PAGE) in a 5–10% Tris-HCl gel, proteins were transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore). The membranes were blocked with 5% non-fat milk in TBST for 1 hour and incubated overnight with mouse monoclonal anti-HA antibody (1:2,500; cwbiotech) or mouse monoclonal anti-GAPDH antibody (1:15,000; cwbiotech). After washing 3 times with TBST, the membranes were incubated with horseradish peroxidase (HRP)-labeled Goat Anti-Mouse IgG (H+L) antibody (1:20,000; KPL) for 1 hour, and developed using Immobilon Western Chemiluminescent HRP Substrate (Millipore).