Immunoprecipitation and dephosphorylation experiments of extrinsic thylakoid polypeptides.

<p>Extraction of extrinsic thylakoid proteins was accomplished by washing the thylakoid membranes with a medium containing low concentrations (0.05%) of Triton X-100. (<b>A</b>) The 30-kDa extrinsic phosphoprotein immunoprecipitates with Cah3. Extrinsic thylakoid proteins released from thylakoid membranes of <i>C. reinhardtii</i> cells acclimated to low CO₂ for 2 h (C) were immunoprecipitated with affinity-purified antibodies against Cah3 and protein A-Sepharose CL-4-B beads. The Sepharose beads were washed and the immunoprecipitate (I) and the supernatant (SN) obtained after centrifugation were analysed by SDS-PAGE and immunoblot and probed with antibodies against Cah3 (left) and Thr(P) (right). (<b>B</b>) Effect of Alkaline phosphatase (AP) treatment on extrinsic proteins released from thylakoid membranes isolated from both high-CO₂-grown cells (High) or cells acclimated to low CO₂ for 2 h (Low). All lanes were loaded with 10 µg protein.