FIGURE 5.

Flot-1 appears not to be required for site-specific GR phosphorylation. LβT2 cells were transfected with NSC or specific mouse Flot-1 siRNA at a final concentration of 40 nm and incubated for 72 h. The cells were incubated for 30 min in serum-free medium with 100 nm Dex or 100 nm GnRH or a combination of both, and the proteins were harvested. The samples were loaded on an 8% SDS-PAGE gel, transferred onto nitrocellulose membrane, and probed with anti-phospho-Ser-212 (P-S212) GR-, anti-phospho-Ser-220 (P-S220) GR-, and anti-phospho Ser-234 (P-S234)-specific antibodies. The membranes were stripped and probed with specific antibodies to GR, Flot-1, and GAPDH. The panels on the left (A, C, and E) show a single representative Western blot, and the graphs (B, D, and F) show the combined results of four independent experiments where vehicle (Ctrl, control) was set to 1. Panel G shows the average percentage decrease of Flot-1 protein for the control samples in the presence of Flot-1 siRNA. One-way ANOVA with Dunnett's post test was used for statistical analysis and is denoted as *, **, or *** to indicate p < 0.05, p < 0.01, or p < 0.001 respectively.