Immunoblotting Protocol for Protein Detection

Immunoblotting was performed as previously described [28 (link),80 (link),81 (link)]. Whole-cell lysates samples (20 μg) or cell fractions (12 μg) were mixed with sample buffer (0.125M Tris/HCl pH6.8, 10% glycerol, 4% SDS, 0.25M DTT) and heated for 5 min at 95 °C. Samples were separated in 12% hand casted polyacrylamide gels using protein electrophoresis (Bio-Rad, Hercules, CA, USA). Separated proteins were blotted onto 0.2 μm nitrocellulose membranes PROTRAN BA 83 (Whatman-Schleicher and Schuell, Maidstone, UK) at 0.25 A for 3 h using a MiniProtean II blotting apparatus (Bio-Rad, Hercules, CA, USA). The membrane was blocked with 5% BSA in TBS for 30min and incubated with the primary antibody at 4 °C overnight. Membranes were washed three times with TBS containing 0.1% Tween-20 (TBS-T). Then the membranes were incubated for 2 h with the corresponding horseradish peroxidase-conjugated secondary antibody. The membrane was again washed three times with TBS-T. The chemiluminescence signal was detected using SuperSignal^TM West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific, Waltham, MA, USA) and a CCD camera GEL Logic 4000 Pro (Carestream Health, New Haven, CT, USA).