Figure 1. ePAB is dynamically phosphorylated during oocyte maturation.

(A) Immature oocyte lysates were untreated (Lysate), or treated with calf alkaline phosphatase (CIP) or CIP in the presence of inhibitors (CIP + PI) and immunoblotted for ePAB. Hyperphosphorylated (Hyper) and hypophosphorylated (Hypo) ePAB are indicated. SDS/PAGE was run under modified conditions (8 % gel, extended electrophoresis time) compared with studies that did not report multiple ePAB electrophoretic forms [6,8,9]. (B) Immunoblots of 2D SDS/PAGE of ePAB from immature and mature oocyte lysates. The pI range is indicated. (C) Mature oocyte lysates were treated with shrimp alkaline phosphatase (Phosphatase) or heat-inactivated phosphatase (Mock) prior to analysis as in (B). (D) Immature or mature oocyte lysates were immunoblotted for ePAB to resolve hyperphosphorylated (Hyper) and hypophosphorylated (Hypo) forms, after CIP treatment where indicated. (E) Schematic showing the phosphorylations identified at Ser⁴⁶⁰, Ser⁴⁶¹, Ser⁴⁶⁴ and Thr⁴⁶⁵ (upper-case bold letters) in the proline-rich region between the RRMs (RNA recognition motifs) and the PABC (PABP C-terminal domain) domain of ePAB, which were mutated to alanine residues (upper-case letters) in the present study.