Figure 6.

GnRH activates MSK, which is required for S28p and also K27ac. A–D, Western blot analysis of nuclear extracts from (A and B) αT3-1 cells or (C and D) LβT2 cells exposed to (A and C) GnRH or (B and D) PMA was carried out using antibody for MSK1p and RNAPII as loading control, while different aliquots of the same sample were analyzed similarly for total MSK. Ratios of the MSK1p:MSK, each after normalization to RNAPII, are reported (A and D) under the gel, or (B and C) mean averages (±S.E.) shown graphically on the right of the gel; t test compared mean values with untreated controls, as described in the legend to Fig. 3. E, similarly, Western blot analysis was carried out for H3S28p, or using antibody to CREBp, which recognizes also ATF1p, in αT3-1 cells treated with various doses of the MSK1 inhibitor (MSKi), RMM-64 for 6 h with H3, RNAPII, or CREB as loading controls. Normalized ratios are shown graphically below. F, αT3-1 cells were serum-starved overnight prior to treatment with GnRH for 6 h and/or RMM-64, added 2 h beforehand. All cells were harvested at the same time, the histone fraction was isolated and Western blot analysis was carried out as described in the legend to Fig. 3; with normalized average (±S.E.) values relative to the untreated control shown graphically at the top right of the blot. G, cells were transfected with shRNA targeting MSK1 or control RNA and levels of H3S28p, H3K27ac, and H3 were assessed by Western blot analysis, as before or RNA extracted for qPCR for Msk1 and Cga; data were analyzed and presented as before. **, p < 0.01; ***, p < 0.001.