Western blot

Total protein from cell lines was extracted in RIPA lysis buffer, and then size-fractionated using SDS polyacrylamide gel electrophoresis. After that, protein was transferred to NC membrane. After blocking, the membrane was incubated with specific testing antibodies for HCA587/MAGE-C2, MAGE-A1 (Bioworld, USA), or MAGE-A3 (Origene, USA) and GAPDH (Bioworld). After blotting overnight at 4ºC, the membrane was incubated with HRP-conjugated anti-mouse or anti-rabbit IgG antibodies for 1h at room temperature. The expression of proteins was then tested by ECL system. GAPDH was used as internal reference.