Western blot analysis and Coomassie Brilliant Blue (CBB) staining

Samples were boiled in sample loading buffer at 100 °C for 10 min, separated by SDS-PAGE, and transferred onto NC membranes for blotting. Membrane blocking was performed for 1 h with 5% skim milk in TBST at RT. After blocking, the membranes were incubated with mouse anti-GST (Santa Cruz Biotechnology), TG2⁷, and FLAG antibodies (Sigma-Aldrich) at 4 °C overnight. The membranes were washed with TBST three times each for 10 min. Primary antibodies were detected with anti-rabbit HRP-conjugated secondary antibody (1:1000, Jackson Laboratory) or anti-mouse HRP-conjugated secondary antibody (1:1000, Pierce). The samples were incubated for 1 h at RT, washed three times with TBST, reacted with Supersignal West Pico solution (Pierce) for 5 min, and exposed to X-ray film (Agfa). For CBB staining, SDS-PAGE gels were stained for 10 min using staining solution (0.1% Coomassie Brilliant Blue G250, 10% acetic acid, 50% methanol, and 40% H₂O₂) and destained with distilled water overnight.