Fig. 2.

JNK Regulates Phosphorylation of the S246 Residue on GR in Vivo

A, COS-7 cells were transfected with pcDNA3 (lanes 1 and 2), pcDNA3-WTGR (lanes 3–8), or pcDNA3-S246AGR (lanes 9 and 10) in the presence of JNK1α1/MLK3 (lanes 5–10). Cells were treated with vehicle (lanes 1, 3, 5, 7, and 9), 100 nm Dex for 1 h (lanes 2, 4, 6, 8, and 10), or 10 μm JNK inhibitor SP600125 30 min before hormone treatment, cell extracts were resolved by SDS-PAGE, and Western blots were probed with antibody specific for phosphorylated S246, total GR, and actin for loading control. B, Quantitative analysis of the GR phosphorylation. Graph represents intensity of the bands corresponding to the GR phosphorylated on the S246 vs. total GR normalized to the actin values shown in panel A. C, H4 cells were treated with vehicle (lane 1), 100 nm Dex (lanes 2–5), 100 nm RU486 (lanes 6–8), and UV (60 J/m²) (lanes 2–8). D, Quantitative analysis of the GR phosphorylation. Graph represents intensity of the bands corresponding to the GR phosphorylated on the S246 vs. total GR normalized to the actin values shown in panel C. E, The same as in panel C except that UV treatment was omitted. Equal amounts of total cellular proteins were analyzed by Western blotting. Immunoblots were probed with antibody specific for phosphorylated S246 (S246-P), GR-specific antibody, and actin antibody. F, H4 cells were incubated in the absence of hormone and treated with UV, and cellular extracts were made as described in panel C. Western blot was stained with Ponceau dye, cut along dotted lines, and strips were probed with antibody specific for phosphorylated S246 in the absence (lanes 1 and 2) or presence of specific peptide used as antigen for this antibody (lane 3, pep). Strips were incubated with preimmune serum (lane 4, PI) or with nonspecific IgG (lane 5, NS) (top panel). Blot was stripped of primary antibodies and probed with GR-specific antibody (middle panel). Lower part of this blot was probed with actin antibody.