Fig. 4

PELP1 is a novel interacting protein of SETDB1. A Mapping of PELP1-SETDB1 interaction region using yeast two hybrid assay. GBD fusions of PELP1 fragments were used to determine the PELP1 binding region in SETDB1. Positive interactors were selected on agar plates lacking either leucine and tryptophan (-LT) or adenine, histidine, leucine, and tryptophan (-AHLT). B, C Total lysates from ZR75 and MCF7 cells cultured in 10% serum were subjected to immunoprecipitation using PELP1 (B) or SETDB1 (C) antibody. IgG antibody was used as a negative control. SETDB1 and PELP1 interaction was confirmed by Western blotting. D ZR75 and MCF7 cells cultured in 10% serum were transfected with vector or GFP-tagged PELP1. The interaction of GFP-tagged PELP1 with SETDB1 was analyzed by immunoprecipitation using GFP-TRAP beads. E, F Total cell lysates from MCF7 cells transfected with T7-PELP1cyto or ZR75 cells transfected with mCherry-PELP1cyto were cultured in 5% DCC medium for 48 h, stimulated with E2 for 15 min, and then subjected to immunoprecipitation using epitope tag antibodies. Interaction between cytoplasmic PELP1 and SETDB1 was analyzed by Western blotting. G Total lysates from ZR75 cells cultured in 10% serum were subjected to GST pull-down assays using the purified GST vector or GST-PELP1 full length proteins. SETDB1 and PELP1 binding was analyzed by Western blotting