Ab209667

The cell proteins were extracted through The Regulation of Investigatory Powers Act (RIPA) lysis buffer (Beyotime) and quantified using bicinchoninic acid (BCA) kit (Beyotime), then 20 μg of proteins were treated by Sodium Dodecyl Sulphate - PolyAcrylamide Gel Electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes (Invitrogen, Eugene, Oregon). Electrochemiluminescent detection system (Thermo Fisher Scientific, Waltham, Massachusetts) was used for blot signal detection. Specific proteins were detected by primary antibody rabbit anti-MAGEC2 (MAGEE1; #ab209667, 1:5000; Abcam, Cambridge, Massachusetts). Anti-GAPDH (#ab9485, 1:2500; Abcam) was used as the internal reference, and HRP-conjugated goat-anti-rabbit IgG (#ab6721, 1:10000; Abcam) was used as secondary antibody.

MAGE-A3/C2 protein staining was performed using 4-µm-thick sections of FFPE tissues. Sections were treated with 3% H₂O₂ and 5% bovine serum albumin for 30 min at room temperature, and then incubated with anti-human MAGE-A3 (1:150 dilution; catalog no. ab140678; Abcam, Cambridge, UK) and MAGE-C2 (1:400 dilution; catalog no. ab209667, Abcam) primary antibodies overnight at 4°C. Following incubation with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature, the sections were washed and counterstained with hematoxylin and visualized under a microscope (Olympus, Tokyo, Japan). Samples were considered positive for MAGE-A3/C2 following the detection of any nuclear and/or cytoplasmic antibody signal in the tumor cells. Samples with complete absence of antibody signal were considered negative for each MAGE tested. Detection of MAGE-A3/C2 expression in testis tissue was used as a positive control and samples that underwent the same process with the absence of primary antibody treatment were used as a negative control.