Figure 2.

Western immunoblot analysis of wild-type and mutated forms of EC-mZP3. Stably transfected cell lines were cultured overnight in serum-free medium, and concentrated culture medium was subjected to HPLC purification on a size-exclusion column. Fractions containing EC-mZP3 were identified by Western immunoblotting by using a goat polyclonal antiserum directed against mZP3 (anti-mZP3). These fractions were pooled, dialyzed, and quantitated by Western immunoassays and by spectrophotometry. Shown are HPLC-purified EC-mZP3 samples (≈200–300 ng) probed with anti-mZP3 and stained as described. Lanes: a, size standards; b, EC-mZP3-[wt]; c, EC-mZP3-[Ser-329]; d, EC-mZP3-[Ser-331]; e, EC-mZP3-[Ser-332]; f, EC-mZP3-[Ser-333]; g, EC-mZP3-[Ser-334]; h, EC-mZP3-[Ser-332–334]; i, EC-mZP3-[Ser-329–334]; and j, EC-mZP3-[Asn-330].