Targeted and restricted complement activation on acrosome-reacted spermatozoa
J. Clin. Invest. Rebecca C. Riley-Vargas, et al. 115:1241 doi:10.1172/JCI23213 [Go to this article.]

Figure 5
Analysis of CRP binding to AR spermatozoa. (A) Western blot of human spermatozoa. Ionophore-treated or untreated spermatozoa were exposed to either 10% normal or EDTA-treated autologous human serum. Lysates were separated in reducing conditions by SDS-PAGE on a 10–20% Tris-glycine gel. The primary antibody used was polyclonal goat anti–human CRP at 1:1,000 dilution. CRP is a pentamer composed of five 25-kDa subunits. (B) FACS analysis of CRP on spermatozoa. Ionophore-treated spermatozoa were exposed to either 10% normal or heat-inactivated autologous human serum. The primary antibody used was polyclonal goat anti–human CRP at 1:1,000 dilution. The secondary antibody used was FITC-linked rabbit anti-goat at 1:10,000 dilution. (C) FACS analysis of spermatozoa exposed to CRP-absorbed serum. Serum was preincubated with immobilized phosphorylcholine to absorb CRP. Spermatozoa were then exposed to 10% normal serum, 10% CRP-absorbed serum, or EDTA-treated human serum. Complement activation was measured by C3d fluorescence of viable AR spermatozoa.