Figure 4.

The involvement of bHLH and PAS domains in the differential recruitment of SRC coactivators. (A) Schematic diagram of the generation of subGRIP1 mutations. (bHLH) Basic helix-loop-helix; (PAS) Per-Arnt-Sim; (NR box) nuclear receptor-binding motifs; (CoRNR box) corepressor nuclear receptor-binding motifs. (B) The effect of sub-GRIP1 mutations on their coactivation of c-Myc and EBAG9 gene expression in T-47D cells after treatment with 1 μM 4-hydroxytamoxifen (Sigma). T-47D cells were seeded in DMEM medium supplemented with 10% FBS. Twenty-four hours later, cells were transfected with either vector, subGRIP1, subGRIP1ΔbHLH, subGRIP1ΔPAS, or subGRIP1ΔbHLH + PAS for 5 h before switching to phenol red-free medium. Forty-eight hours after transfection, cells were collected and total RNA was extracted using TRIZOL Reagent. Expression of c-Myc and EBAG9 mRNA was measured by real-time RT-PCR. (C) ChIP analysis of SRC coactivator recruitment in subGRIP1 and its mutants-transfected T-47D cells. T-47D cells were seeded in DMEM medium supplemented with 10% FBS. Twenty-four hours later, cells were transfected with either vector, subGRIP1, subGRIP1ΔbHLH, subGRIP1ΔPAS, or subGRIP1ΔbHLH + PAS for 5 h before switching to phenol red-free medium. Forty-eight hours after transfection, ChIP assays were performed using specific antibodies against GRIP1 and AIB1. (D) Western blotting analysis of the protein expression. T-47D cells were seeded in DMEM medium supplemented with 10% FBS. Twenty-four hours later, cells were transfected with either vector, sub-GRIP1, subGRIP1ΔbHLH, subGRIP1ΔPAS, or subGRIP1ΔbHLH + PAS for 5 h before switching to phenol red-free medium. Forty-eight hours after transfection, cells were collected, and cellular proteins were extracted for Western blotting analysis with antibodies against GRIP1. Transfection efficiency was monitored by cotransfection with an E. coli lacZ construct (pcDNA4/His/Max/lacZ, Invitrogen Corp.).