Figure 4.

The CC Domains of PHB and REA Are Required for Hetero-Oligomerization, Stabilization, and Transcriptional Cross-Squelching

A, Schematic diagram of functional domains of PHB and REA. B, Transient expression of carboxyl-terminal V5 or flag tagged PHB and REA wild-type and CC deletion mutants in 293T cells. Protein extracts were first immunoprecipitated (IP) with anti-flag antibody, and precipitated protein complexes were analyzed by Western blot (WB) analysis with an anti-V5 antibody. Bands in lanes 1–5 in B-a represent the immunoprecipitated V5-tagged PHB, whereas bands in lanes 6–10 represent the immunoprecipitated V5-tagged REA. B-b through -d, Input control of different tagged PHB and REA wild type and mutants and β-actin. C, REA-V5, PHBV5, REA-flag, and PHB-flag were overexpressed in 293T cells separately. D, Flag-tagged PHB and REA wild types or CC deletion mutants were expressed in 293T cells. The protein synthesis inhibitor cycloheximide (CHX) was added to a final concentration of 200 μg/ml and cells were harvested at the indicated time points. Protein stability was measured by Western blot analysis with anti-flag antibody (top panels of D-a through -d), and anti-β-actin antibody as an input control (bottom panels of D-a through -d). E, The CC domains are required for the cross-squelching actions between PHB and REA. The HepG2 cells were cotransfected with ERE-luc reporter vector, expression vectors of ERα (5 ng), REAΔCC (300 ng), PHBΔCC (300 ng), REAΔ/PHBΔ (150 ng of REAΔCC and 150 ng of PHBΔCC), REA (100 ng), PHB (100 ng), REA/PHB (50 ng of REA and 50 ng of PHB), REA (300 ng), PHB (300 ng), or REA/PHB (150 ng of REA and 150 ng of PHB). F, V5-HDAC1, PHB-flag, and PHBΔCC were overexpressed in 293T cells separately. Western blot analysis was used to determine the amounts of cell lysates that contain equal levels of PHB wild type and PHBΔCC. After this, the cell lysates containing either contain PHB-flag or PHBΔCC-flag was mixed with equal amounts of cell lysates containing V5-HDAC1. After overnight incubation, the cell lysates were immunoprecipitated with beads linked to anti-flag antibodies, and probed with antibodies against V5.