2.4. Western Blot

SDS-PAGE (8–12%) resolving gel was used for Western blot. A total of 30 µg protein was separated and allowed to transfer onto polyvinylidene fluoride (PVDF) membrane for 90 min at 200 mA at 4 °C [24]. Non-specific sites were blocked using 5% fat-free skimmed milk for 1 h at room temperature (RT). PVDF membranes were probed with specific antibodies (Santa Cruz Biotechnology, Paso Robles, CA, USA or Cell Signaling, Beverly, MA, USA) for overnight (12 h) at 4 °C. Next day, horseradish peroxidase (HRP)-conjugated secondary antibodies were used for 1 h at RT. Finally, membranes were washed with Tris-buffer saline (TBS) containing 0.1% Tween 20 (T20) and 1% BSA and bands were visualized using enhanced chemiluminescence (ECL) kit.