Western blotting by ECL detection: cell lysate proteins were separated by electrophoresis on a 4-12% Bis-Tris gel (Nupage, Invitrogen) in MES/SDS buffer at 150 V for 70 min before transfer onto nitrocellulose membranes (GE Healthcare). Non-specific antibody binding was blocked by incubating the membranes for 1 h in TBS with 0.1% Tween20 (TBST) containing 5% skimmed milk powder. Membrane strips were then incubated with primary antibodies diluted in TBST/milk for 1-2 h at room temperature. After three washes with TBST, membranes were incubated with HRP-conjugated secondary antibodies (Sigma) in TBST/milk for 1 h. After a further three washes with TBST, membranes were treated for 1 min with peroxide and Luminol reagents (Millipore, Billerica, MA) before imaging using a Bio-Rad ChemiDoc Imager.
