FIG. 5.
Sp110b is a potent transcriptional corepressor of RARα. (A through C) Activation and suppression of RARα-mediated transcription by Sp110 and Sp110b, respectively. COS-7 cells were transfected with 25 ng of pRARE-Luc with pCA-Sp110 (A), pCMV-Sp110b (B), or pCMV-Sp110b with pSG5-RARα (C) at the indicated doses for a total of 400 ng of plasmids by adjusting with the empty vector. Additional conditions were as described in the legend to Fig. 2A. RARα protein production levels for each condition were examined by immunoblot analysis (C, lower panel). (D) Interaction of Sp110 and Sp110b with RARα. A GST pulldown assay was performed as described in Materials and Methods. 35S-labeled RARα was incubated with GST-Sp110, GST-Sp110b, or GST in the absence [ATRA(−)] or presence [ATRA (+)] of 1 μM ATRA. The Coomassie brilliant blue (CBB) staining pattern of pulled-down products is shown in the bottom panel. Positions of molecular size markers are given on the left. Arrowhead, circle, and square indicate bands corresponding to GST, GST-Sp110, and GST-Sp110b, respectively. (E) Coimmunoprecipitation assay detecting the interaction between Sp110 and RARα. Lysates from ATRA-treated 293T cells overproducing FLAG-tagged Sp110b (FL-Sp110b) and/or RXRα with HA-tagged RARα (HA-RARα) were used for coimmunoprecipitation followed by immunoblot analysis. Data are presented essentially as described in the legend to Fig. 3C. IgG, normal mouse IgG; HA, anti-HA antibody. FL-Sp110b coimmunoprecipitated (co-IP) with HA-RARα was detected by using an anti-FLAG antibody (upper panel). Center and lower panels show HA-RARα and FL-Sp110b in total-cell lysates detected by anti-HA and anti-FLAG antibodies, respectively. (F) DNA-protein complex immunoprecipitation assay detecting the RARα-Sp110b-RARE complex. 293T cells overproducing either RXRα with HA-RARα or FL-Sp110b, or both, and carrying either pRARE-Luc (RARE +) or the p-55BLuc reporter plasmid lacking RARE (RARE −), were treated with ATRA. Formaldehyde-cross-linked DNA-protein complexes were then immunoprecipitated with anti-HA (HA), anti-FLAG (FL), or normal mouse IgG (IgG). The DNA extracted from the respective immunoprecipitates was amplified by PCR as described in Materials and Methods.