Fig 1. Detection of Cip29 and phosphorylated Cip29 proteins in X. laevis egg extract.

(A) Immunodetection of Cip29 on a Western blot of X. laevis egg extract (E), in vitro translated Cip29 (T1) and in vitro translated Cip29b (T2). A non-specific band detected by the α-Cip29 antibodies in X. laevis egg extract is indicated with an asterisk. Molecular weight markers (kDa) are indicated on the left. (B) Western blot of Cip29 in X. laevis egg extract, undepleted or immunodepleted with either non-specific rabbit IgGs (mock) or α-Cip29 antibodies (left panel), and in the immunoprecipitated samples (right panel). The non-specific band (*) serves as a loading control for extract samples. (C) Amino acid sequence alignment of X. laevis Cip29 and Cip29b. (D) Western blots of egg extract incubated with or without 50ng/μl AT₇₀ (21^°C, 1h). Samples were resolved by electrophoresis on a standard 12% SDS-PAGE gel (left panel) and an 8% SDS-PAGE gel containing 15μM Phos-tag (right panel) before immunoblotting with the indicated antibodies. Modified Cip29 protein is denoted by Cip29^-P (E) Cip29 was immunoprecipitated from egg extract supplemented with 50ng/μl AT₇₀ (21^°C, 1h). The immunoprecipitation reactions were treated with either 1 x NEBuffer 3 (-CIP), or NEBuffer 3 containing 10 units of CIP, or 10 units of heat-inactivated CIP (21^°C, 1h) before glycine elution of the immunoprecipitated proteins. Eluted protein was supplemented with 2μg of Cip29-depleted extract before separation on Phos-tag SDS-PAGE, to optimise resolution of the modified form of Cip29.