FIG. 5.

A) Immunoblotted egg and oocyte extracts probed with anti-YWHA and reprobed with anti-p(S)YWHA binding motif antibody, indicating the presence of the YWHA protein in both egg and oocyte. Confirming the previous experiments, the phosphorylated YWHA binding motif of PADI6 is recognized only in egg extracts. B—D) YWHA pull-down assays in eggs and oocytes. Triton X-100-extracted eggs or oocytes were incubated with GST-YWHA fusion protein attached to glutathione sepharose beads (lanes labeled GST-YWHA). In the control experiments, extracts were incubated with recombinant GST alone attached to the beads (lanes labeled GST alone or GST control). B) GST-YWHA bound a protein in egg extracts that was labeled with the anti-p(S)YWHA binding motif antibody (GST-YWHA, Bound lane). GST alone did not bind this protein (GST alone, Bound lane) but it was present in the flow-through or eluate (GST alone, Bound lane). C) A similar GST-YWHA pull-down experiment was performed egg extracts. Following polyacrylamide gel electrophoresis, the proteins were blotted and probed with anti-PADI6; GST-YWHA bound PADI6 (GST-YWHA, Bound lane). As expected, not all of the PADI6 was bound, and some could be detected in the eluate (GST-YWHA, Unbound lane). In control experiments with egg preparations, GST alone did not bind PADI6 (GST Control, Bound lane), but it was detected in the eluate (GST Control, Unbound). D) GST-YWHA did not bind PADI6 in oocyte extracts (GST-YWHA, Bound lane), nor did GST alone (GST Control, Bound), though PADI6 was in the eluate (Unbound lanes). B–D are representative of three separate experiments.