Mini blot

Forty micrograms (40 µg) of cytoplasmic, 20 µg of nuclear and 15 µg of histone protein extracts were separated by SDS-polyacrylamide gels and transferred electrophoretically onto PVDF membranes (either 0.45 or 0.2 µm) using the mini-gel tank and mini-blot modules from Invitrogen (Carlsbad, CA, USA), respectively. The blots were then blocked in 5% non-fat milk powder in TBST buffer (50 mM Tris–HCl, 150 mM NaCl at pH 7.6 and 0.1% Tween-20) for 2 h at room temperature. After blocking, membranes were washed three times with TBST and incubated overnight at 4 °C, under agitation, with the appropriate primary antibody and according to the manufacturer’s protocol. Next day, membranes were incubated with the appropriate horseradish peroxidase-conjugated secondary antibody (mouse or rabbit at 1:1000) for 1 h at room temperature, under agitation, after being washed three times with TBST. After incubation with the secondary antibody, membranes were washed three times with TBST and labeled protein bands were detected by utilizing the SuperSignal West Pico PLUS Chemiluminescent Substrate from Thermo Scientific (Waltham, MA, USA) according to the manufacturer’s protocol. Protein bands were visualized with the use of the G:BOX Chemi XX6/XX9 gel imaging system (Syngene, Cambridge, UK).