Western blot

Sucrose fractions were separated by SDS-PAGE on precast 10% Bis-Tris gels with MES running buffer or 4–12% gradient Bis-Tris gels with MOPS running buffer (NuPage, Invitrogen). Proteins were then transferred to 0.2 µm pore-size nitrocellulose membrane and blocked with 5% non-fat dry milk powder in TBS buffer containing 20 mM Tris, 0.15 M NaCl, and 0.05% Tween 20, pH 7.4 for 2 h at 20 ± 2 °C. For quantitative western blots protein concentrations in sucrose fractions were measured using Pierce660 reagent (Sigma Aldrich). The immunoblots were analyzed with anti-alpha 1 Na⁺/K⁺-ATPase, anti-SDHA, anti-alpha tubulin, anti-perforin, anti-β actin, anti-synaptobrevin2, anti-RFP, anti-granzyme B, anti-RPS10, anti-Sec61β, NFAT1 and horseradish peroxidase (HRP)-conjugated goat anti-mouse (H + L or light chain for immunoprecipitation) or anti-rabbit secondary antibodies (F(ab’)2, heavy or light chain) (Table 1). For reprobing western blot membranes were stripped in stripping solution (Invitrogen) for 5–15 min at 20 ± 2 °C. Finally, the blot was developed using enhanced chemoluminescence reagents (SuperSignal West Dura Chemoluminescent Substrate; Thermo Fisher Scientific) and imaged by gel documentation (FluorChem M system, ProteinSimple).