Fig. 4.

CGRP evokes increases in the expressions of EZH2 and H3K27me3 in microglia by PKA/PKC. a, b The expression of Iba1 (a marker of microglia, red) and its colocalization with CRLR, RAMP1, or CRCP staining (green) in cultured BV2 cells a and rat primary microglia b. Scale bar 40 μm. c, d Western blot analyses of EZH2 and H3K27me3 expressions in BV2 cells c and rat primary microglia d with treatment of CGRP at 0, 1, 2, 4, 6, and 12 h, respectively. e Western blot analyses for EZH2 and H3K27me3 protein levels in BV2 microglial cells with co-treatment of CGRP (1 μM) and GSK126 (5 nM) for 4 h. f Western blotting analyses for EZH2 and H3K27me3 protein levels in microglial cells (BV2) with treatment of CGRP peptide (1 μM) for 4 h and pretreatment with 1 μM forskolin (PKA activator), 3 μM PKI6-22 (PKA inhibitor), 325 nM PMA (PKC activator), or 5 μM chelerythrine chloride (PKC inhibitor) for 30 min. The mean optic densities of the proteins were calculated by normalizing to GAPDH. All values are expressed as the means ± SEMs (n = 4).^*p < 0.05 vs. controls, ^#p < 0.05 vs. CGRP only groups. g Shown is an example of microglial cell growth curves by RTCA. RTCA was performed to evaluate the proliferation and viability of microglial cells with continuous treatment of CGRP and co-treatment of GSK126