Analysis of GCAP2 phosphorylation by isoelectrofocusing.

<p><b>A</b>. Isoelectrofocusing (IEF) gel of light-adapted wildtype mouse retinal homogenates. Mice were light-adapted to room light. Retinas were obtained, solubilized in a saline buffer with 1% dodecyl maltoside, in the presence or absence of 50 mM NaF (phosphatase inhibitor). Samples were clarified, loaded onto an electrofocusing gel (pH range 3–8) and focused for 2 h at 23W. Proteins were transferred to a nitrocellulose membrane and incubated with an anti-GCAP2 Ab. Two prominent bands are observed at 4.92 and 4.85 isoelectric point, that correspond to unphosphorylated and monophosphorylated mGCAP2, respectively. <b>B</b>. The overall phosphorylation status of GCAP2 does not change significantly during a 12 h period of dark-adaptation. Mice were light-adapted to room light for 1 h, and subjected to dark-adaptation for a period of up to 14 h. Retinas were analyzed as above. <b>C</b>. Analysis of GCAP2 phosphorylation status in the indicated mouse lines. Transgenic bGCAP2 is phosphorylated to a lesser extent than the endogenous mGCAP2, whereas bEF⁻GCAP2 is phosphorylated to a much higher extent. Note that the isoelectric point of bGCAP2 differs from that of mGCAP2, and that the isoelectric point of bEF⁻GCAP2 (E80Q,E116Q,D158N GCAP2) is shifted versus that of bGCAP2. Results from the isoelectrofocusing gels confirm that transgenic bEF⁻GCAP2 is phosphorylated to a much higher extent than the control transgenic bGCAP2.