Mage a4

For immunoblot analysis, cells were lysed in RIPA and whole cell lysates resolved by 10% SDS-PAGE and transferred to PVDF membrane. Immunoblots were probed using the following antibodies where specified: MAGE-A3 IgG1-kappa monoclonal antibody (Proteintech 60054-1-Ig), MAGE-A4 (Cell Signaling (E7O1U) XP Rabbit mAb #82491), p53 (Cell Signaling (1C12) Mouse mAb #2524), Actin IgG1-kappa monoclonal antibody (Thermo Fisher #MA5-11869), GAPDH (Cell Signaling 14C10) and cyclin antibodies (Cell Signaling: Cyclin A2 (BF683), Cyclin B1 (4138), Cyclin D1 (92G2), Cyclin D2 (D52F9), Cyclin D3 (DCS22), and Cyclin E1 (HE12)). Anti-mouse (Cell Signaling 7076s) and anti-rabbit (Cell Signaling 7074s) HRP-conjugated secondary antibodies were used for detection using HRP detection reagent (Thermo Scientific product # 1859698) and autoradiography film (Denville Scientific cat # E3012).

Cultured tissue cells were lysed in lysis buffer supplemented with a protease inhibitor cocktail and disrupted on ice using a sonicator. The protein concentration of cell lysates was assessed using the Bradford protein assay (Bio-Rad Laboratories, Hercules, CA, USA) with bovine serum albumin as a standard. Each cell lysate (20 µg) was subjected to SDS-PAGE using a 5-20% gel (Wako, Osaka, Japan) and transferred to a PVDF membrane. After blocking with PVDF-blocking reagent (Toyobo, Osaka, Japan), 1 μg/mL of purified polyclonal antibody for each antigen in Can Get Signal 1 (Toyobo) were incubated with the membrane. Immunoreactive antigens were detected using anti-rabbit IgG HRP-linked antibody (Cell Signaling Technology, Tokyo, Japan) and Western Lightning Plus ECL (PerkinElmer, Waltham, MA, USA). The control monoclonal antibodies to MAGE-A4 (clone: E701U, Cell Signaling Technology, Tokyo, Japan) and XAGE-1b (clone: USO9-13) (47 (link)) were employed for the validation of the specificity of polyclonal antibodies. The membrane was reprobed with an anti-β-tubulin antibody (Wako).