Figure 1.

Detection of endogenous SMRT proteins. HeLa nuclear extract, together with in vitro-translated [³⁵S]methionine-labeled N-CoR and C-SMRT, were separated on a SDS/PAGE. The N-CoR and C-SMRT polypeptides were detected by autoradiography (Left). An identical gel was processed for Western blotting using an affinity-purified rabbit anti-C-SMRT polyclonal antibody and detected by 5-bromo-4-chloro-3-indolyl phosphate (BCIP)/nitroblue tetrazolium (NBT) color reaction (Center). One major polypeptide similar to the size of N-CoR (270 kDa) was detected in the HeLa nuclear extract, in addition to two minor bands of 180 and 80 kDa, respectively (arrows). The anti-SMRT antibody does not crossreact with N-CoR. The same HeLa nuclear extract also was processed for Western blotting using anti-C-SMRT antibody but developed by ECL⁺ reaction (Right). The three specific SMRT polypeptides and two nonspecific bands (open arrowheads) below 80 kDa were indicated.