Fig. 1

ERα phosphorylated at Ser216 in mouse brain microglia. a Microglia. Mixed glia cells were prepared, cultured for 4-days and double-stained with either an anti-ERα (in green) or an anti-P-S216 (in green) antibody and an anti-Iba-1 (in red) antibody for microglia marker and visualized as described in the Methods section. Nuclei were stained by DAPI in blue. b Enriched microglia. Microglia was enriched from glia cells as described in the Methods section for double staining. c Astrocytes. Mixed glia cells were double-stained with either an anti-ERα (in green) or an anti-P-S216 (in green) antibody and an anti-GFAP (in red) antibody for astrocyte marker and were visualized as described in the Methods section. Nuclei were stained by DAPI in blue. d Glia cells obtained from 2-days-old neonates of Ex3-ERα KO and wild type (WT) mice were cultured and double-stained by an anti-ERα or an anti-P-Ser-216 (P-S216) (in green) and an anti-Iba-1 (in red) antibodies and visualized as described in the Methods section. Nuclei were stained by DAPI in blue. e Whole lysates prepared from enriched microglia were subjected to Western blots by an anti-ERα or an anti- P-S216 antibody. An an anti-β-Actin antibody was utilized to verify equal amounts of proteins in an each well