Fig. 6.

Increased interaction of SHP with LRH-1 mediated by 3Cl-AHPC is severely impaired by mutation of Leu-100. A, Experimental outlines. B–D, HepG2 cell extracts were prepared and then treated in vitro with 3Cl-AHPC to avoid the activation of cellular kinase signaling, and then CoIP assays were performed. LRH-1, Brm, or HDAC1 was immunoprecipitated from cell extracts, and presence of flag-SHP was detected by M2 antibody. In all in vitro CoIP protein interaction assays, IgG was used as a negative control. Similar levels of LRH-1, Brm, and HDAC1 in the immunoprecipitated samples were detected by Western blot analysis (WB). Similar levels of the L100E mutant and SHP wild type (WT) in input samples were also confirmed by Western blot analysis. Band intensities were determined using ImageJ, and the values for control samples treated with vehicle were set to 1. Kd, Dissociation constant.