Figure 7. mPGES-1 enhances nuclear β-catenin association with EGR1 and TCF4 in Huh7 cells.

Huh7 cells were transfected with different vectors (mPGES-1 expression or RNAi vector, β-catenin expression vector, GSK-3β expression vector, EGR1 expression or RNAi vector, and control vectors) as indicated on the top of the panels. The nuclear extracts were obtained and subjected to immunoprecipitation by using specific antibodies or IgG as the control, followed by western blotting using specific antibodies as indicated for each panel. The nuclear extracts or whole cell lysates were also processed for routine western blotting analysis. IP – immunoprecipitation; WB – western blotting.

A. mPGES-1 increases the association between β-catenin and EGR1 in the nuclei isolated from Huh7 stable cell lines. Nuclear β-catenin was used as input control for immunoprecipitation.

B. mPGES-1 increases β-catenin interaction with EGR1, TCF4 or LEF1 in Huh7 cell lines. (Upper Panels) Nuclear extracts were obtained from Huh7 cells with or without mPGES-1 overexpression and processed for immunoprecipitation with anti-β-catenin antibody (or IgG as control), followed by western blotting analysis for EGR1 and TCF4. (Mid Panels) The aforementioned first immunoprecipitates were cleansed with the elution buffer (0.1% Triton X-100, 0.1% SDS, 0.5% BSA in PBS) (40μl aliquot first immunoprecipitates was eluted with 750μl elution buffer and incubated for 50min at room temperature). The samples were then subjected to repeat immunoprecipitation (second IP) with anti-LEF1 antibody (or IgG as control), followed by western blotting analysis for EGR1 and β-catenin. (Lower Panels) The aforementioned second immunoprecipitates were cleansed with the elution buffer and subjected to an additional repeat immunoprecipitation (third IP) with anti-TCF4 antibody (or IgG as control), followed by western blotting analysis for EGR1 and β-catenin.

C. mPGES-1 increases β-catenin binding to EGR1, but not SUM-EGR1 in Huh7 stable cell lines. Nuclear β-catenin was used as input control for immunoprecipitation.

D. mPGES-1 increases the interaction between β-catenin and TCF4 in the nuclei isolated from Huh7 stable cell lines; the interaction is enhanced by EGR1 overexpression. Nuclear β-catenin was used as input control for immunoprecipitation.

E. mPGES-1 increases the interaction between β-catenin and LEF1 in the nuclei isolated from Huh7 stable cell lines; the interaction is influenced by EGR1.

F. mPGES-1 increases β-catenin interaction with EGR1 or TCF4 in Huh7 stable cell lines; this interaction is partially inhibited by GSK-3β. GSK-3β overexpression increases EGR1 sumoylation. Western blots for SUMO-EGR1 (102 KD) or EGR1 (82 KD) were used as input controls.