Ny eso 1

Whole-cell proteins were extracted in RIPA buffer with proteasome inhibitor. Protein content was quantified by the BCA assay (Pierce, Rockford, USA). Forty micrograms of whole cell lysates were separated by 10% SDS-PAGE and transferred to PVDF membranes. Membranes were blocked with 5% non-fat dry milk in Tris-bufferd saline with Tween 20 (TBST) for 2 hours and probed with primary antibodies diluted in TBST containing 5% milk at 4°C overnight. Blots were incubated with antibodies against human MAGE-A (6C1; Santa Cruz Biotechnology, Santa Cruz, USA), NY-ESO-1(Santa Cruz Biotechnology) and GAPDH (Abcam, Cambridge, USA). The anti-MAGE-A antibody 6C1 cross-reacts with MAGE-A1, A2, A3, A4, A6, A10 and A12. The molecular weight of MAGE-A10 is 72 kDa, and that of the rest ranges from 45–50 kDa. Membranes were washed and incubated with horseradish peroxidase-conjugated rabbit anti-mouse secondary antibody (Santa Cruz Biotechnology) for 1 hour at room temperature. Targeted proteins were visualized using an enhanced chemiluminescence (ECL) detection system (ChemiDoc™ XRS+ imaging system; BIO-RAD, Hercules, USA) and hyper-ECL film. Band analysis for gray value was performed by the Quantity One software (BIO-RAD).

Immunohistochemical staining for CTA was performed on TMAs using MAGE-A3 (1:500, Santa Cruz Biotechnology, Dallas, TX), SSX2 (1:200, Origene, Rockville, MD), and NY- ESO-1(1:200, Santa Cruz Biotechnology, Dallas, TX), PBK (1:2000, Thermo Scientific, Walthman, MA) and TTK (1:1000, Thermo Scientific, Walthman, MA), SPA17 (1:200, Thermo Scientific, Walthman, MA). Slides were deparaffinized and rehydrated through graded CitriSolv and alcohol solutions. Antigen retrieval was performed by heat-induced epitope retrieval, in which slides were heated in sodium citrate buffer (pH 6.0) in a microwave for 10 min and cooled down to RT for 30 min. Slides were quenched in 3% H₂O₂ for 10 min to block endogenous peroxidase activity. After rinsing with PBS, slides were incubated for 10 min in DAKO protein block serum-free to inhibit non-specific staining. Slides were rinsed in PBS and treated with primary antibodies at 4 °C overnight. ImmPRESS® HRP Anti-Mouse IgG (Peroxidase) Polymer Detection Kit is applied to slides and incubated for 30 min at room temperature. Staining was visualized with DAB (5-min development). Slides were counterstained in Harris Modified hematoxylin and dehydrated through graded ethanol and CitriSolv solutions. Testis tissue was used as a positive control for all cancer testes antigens staining.