2.9. Western blot

Cells were lysed in RIPA buffer (40 mM HEPES, pH 7.4, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 0.5% DOC-Na, 0.1% SDS, 25 mM NaF, 1 mM NaV₃O₄) supplemented with 1× protease inhibitor cocktail (Nakarai Tesque). After centrifugation for 20 min at 26,000 × g and 4°C, aliquots of lysate containing 30 μg of protein were subjected to SDS-polyacrylamide gel electrophoresis, after which the proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Immobilon-P; Merck Millipore). The membranes were then blocked for 30 min at room temperature in Tris-buffered saline (TBS; 10 mM Tris–HCl, pH 7.5 and 150 mM NaCl) containing 0.05% Tween-20 (TBS-T) with 5% non-fat skim milk or 1% BSA in TBS-T for detection of phosphorylated STAT1. Once blocked, the membranes were probed first with specific primary antibodies overnight at 4°C and then with the appropriate HRP-conjugated secondary antibodies (Bio-Rad). Blots were visualized using ECL Prime Western Blotting Detection Reagent (Cytiva) and were captured using an ODYSSEY Fc Imaging System (LI-COR Biosciences). For cell supernatant analysis, the medium was collected, concentrated using Amicon Ultra-0.5 3K and subjected to Western blot as above.