Horseradish peroxidase conjugated secondary anti mouse antibody or anti rabbit antibody

The cells were harvested and lysed in a buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 2 mM EDTA, 1% Triton, 1 mM PMSF and a protease inhibitor cocktail (Sigma-Aldrich) for 20 min on ice. Lysates were separated by centrifugation at 16,000 × g, at 4°C for 10 min. Supernatants were collected and protein concentrations were determined by the Bradford assay (Bio-Rad, Hercules, CA, USA). The proteins were then separated with an SDS/polyacrylamide gel and transferred to a nitrocellulose membrane (Bio-Rad). Subsequent to being blocked in phosphate-buffered saline (PBS) with 5% skimmed dry milk for 1 h, the membranes were incubated overnight at 4–8°C with the primary antibodies in PBS with 5% skimmed dry milk. The following antibodies were utilized: Anti-LDHA rabbit antibody (1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA) and anti-β-actin monoclonal antibody (1:2,000; Sigma-Aldrich). Membranes were extensively washed with PBS and incubated with horseradish peroxidase conjugated secondary anti-mouse antibody or anti-rabbit antibody (1:2,000; Bio-Rad). Subsequent to additional washes with PBS, antigen-antibody complexes were visualized with an enhanced chemiluminescence kit (Pierce Biotechnology, Inc., Rockford, IL, USA).

Cells were harvested and lysed in NETN (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5% NP40) for 20 min on ice. Lysates were cleared by centrifugation at 13 200 r.p.m. at 4 °C for 5 min. Supernatants were collected and protein concentrations were determined by the Bradford assay (Bio-Rad, Hercules, CA, USA). The proteins were then separated with a SDS-polyacrylamide gel and transferred to a nitrocellulose membrane (Bio-Rad). After blocking in TBS with 5% nonfat milk for 1 h, the membranes were incubated overnight at 4 °C with the primary antibodies in TBST with 1% BSA. The following antibodies were purchased from Cell Signaling (Beverly, MA, USA): p-ERK1/2, t-ERK, p-ErbB2, and tubulin. Antibodies were purchased from other companies as follows: β-actin antibody (Sigma), MST4 (Abcam, Cambridge, MA, USA), t-ErbB2 (Calbiochem, Billerica, MA, USA). Membranes were extensively washed with TBST and incubated with horseradish peroxidase conjugated secondary anti-mouse antibody or anti-rabbit antibody (dilution 1 : 2000, Bio-Rad). After additional washes with TBST, antigen–antibody complexes were visualized with the enhanced chemiluminescence kit (Pierce, Rockford, IL, USA).