Figure 4. Phosphorylated ERα in peripheral blood and peritoneal neutrophils.

(A) WBC were isolated from peripheral blood collected from female mice for double staining with an anti-Ly6G antibody (αLy6G) and either an anti-P-Ser-216 (αP-S216) or an anti-ERαN (αERαN) antibody. Staining and nuclei were visualized as described in the legend of Figure 2. (B) Peritoneal neutrophils were prepared on slides as described in the Materials and Methods section and double stained by αLy6G (in red) and that by αP-S216 (in green). This picture overlaps the double antibody staining with DAPI staining. (C) Peritoneal Neutrophils were cultured with ethanol (vehicle) or estradiol (E2, 10 nM) for 15 or 60 min at 37 °C, from which whole extracts were prepared for Western blots with αP-S216, αERαN or anti-β-Actin (αβActin) antibody. Whole extracts that contained recombinant human ERα (not phosphorylated) was utilized as an ERα marker. αβActin staining endogenous actin was utilized to verify equal amounts of proteins in each well.