Figure 5. Regulation of ERα phosphorylation in neutrophils.

(A) WBC fractions were isolated from C3H/HeNCrIBR females and males and sham-operated and ovariectomized C3H/HeNCrIBR females after treatment with vehicle or estradiol (E2). Pictures present triple-stained cells with αLy6G (in red), αP-S216 (in green) antibodies and DAPI (in blue). (B) Levels of phosphorylated ERα-expressing blood neutrophils in C3H/HeNCrIBR, C57BL/6 and their F₁ progeny. WBC fractions were isolated from peripheral blood collected from each of five females for a given group and double stained with αLy6G and either αP-S216 or αERαN. Cells double-stained with αP-S216 or αERαN were counted and graphed as percentages of the total neutrophils stained by αLy6G. These numbers counted for staining with αP-S216 were 2000, 200 and 700 for C3H/HeNCrIBR, C57BL/6 and their F1 progeny, respectively, and 2000, 200 and 700 for staining with αERαN. The percentage of αP-S216 antibody-positive cells to total neutrophils was 18.5 ± 0.6. The corresponding percentages were 7.7 ± 0.4 and 12.6 ± 0.5 in blood neutrophils from C57BL/6 and F1 females, respectively. Those percentages of αERαN-positive neutrophils were nearly identical to those obtained for phosphorylated ERα with αP-S216 antibody: 19.5 ± 1.0, 5.9 ± 0.9 and 12.6 ± 1.2 for C3H/HeNCrIBR, C57BL/6 and F1 progenies, respectively. Significances were P <0.0001 and P=0.0002 between C3H/HeNCrIBR and C57BL/6 and C57BL/6 and F1, respectively, for αP-S216 antibody-positive cells. The corresponding values for αERαN antibody-positive cells were P<0.0001 and P=003 between C3H/HeNCrIBR and C57BL/6 and C57BL/6 and F1, respectively.