Figure 8.
Immunolocalization and pull-down assay of HCF243. A, Immunolocalization of HCF243. The wild-type (WT) thylakoid membranes were sonicated in the presence of 250 mm NaCl, 200 mm Na2CO3, 1 m CaCl2, and 6 m urea for 30 min at 4°C. PsbO (the 33-kD luminal protein of PSII) and CP47 (the PSII core protein) were used as markers. Membranes that had not been subjected to any salt treatment were used as controls. B, Pull-down assay of HCF243 and D1 protein interaction. About 10 μg of HCF243 His tag fusion protein coupled to Ni-NTA resin was incubated with 100 mg of DM-solubilized thylakoid membranes. Bound proteins were eluted, separated by SDS-PAGE, and subjected to immunoblot analysis with D1 and CP47 antibodies. Similar results were obtained in two additional independent experiments.