Recruitment of endogenous transcriptional corepressors by TBLR1-RARα in 293T cells. In Co-IP assays (A-B,D), 293T cells were transfected with Myc-tagged TBLR1-RARα (A[left],B,D[left]) or RARα (A[right],D[right]) expression plasmid, and Flag-tagged TBLR1-RARα expression plasmid in coimmunofluorescence analysis (C,E). (A) Co-IP between endogenous corepressors and TBLR1-RARα. TBLR1-RARα interacted with N-CoR, mSIN3A, HDAC3, H2B, and TBL1 but could not interact with RARα (left). Co-IP between endogenous corepressors and RARα. RARα could associate with N-CoR, SMRT, mSIN3A, and HDAC3, but not with H2B (right). (B) N-CoR was detected to associate with TBLR1-RARα and HDAC3 by Co-IP assays. (C) Coimmunofluorescence analysis of colocalization of Flag-tagged TBLR1-RARα and endogenous corepressors. Flag antibody and antibodies for corepressors were used as primary antibodies, and DAPI was used for nuclear staining. At least 100 cells were counted for colocalization analysis. (D) Co-IP assays between endogenous corepressors and TBLR1-RARα were conducted in the presence of 1 uM ATRA for 48 hours. TBLR1-RARα interacted with N-CoR, SMRT, and HDAC3, but not mSIN3A (left). Co-IP assays between endogenous corepressors and RARα were performed after ATRA treatment. RARα could only coimmunoprecipitate with HDAC3, but not N-CoR, SMRT, and mSIN3A (right). (E) Coimmunofluorescence analysis of colocalization of Flag-tagged TBLR1-RARα and endogenous corepressors was performed in the presence of ATRA. Bars (C,E) represent 8 μm.