Figure 8.

Immunolocalization of HCF244. A, Chloroplast localization of HCF244. Intact chloroplasts were isolated from 5- to 6-week-old wild-type plants by Percoll step gradient centrifugation and fractionated into chloroplast membranes and stroma proteins. An equivalent of 10 μg of chlorophyll was loaded on the gel, together with a total leaf extract containing the same protein amount as the total chloroplast extract. B, Salt washing of thylakoid membranes. Thylakoids were sonificated in the indicated buffers, then afterward separated into membrane and soluble proteins by centrifugation and analyzed by SDS-PAGE. C, Protease protection assay. Intact and sonificated thylakoids were treated with thermolysin (final concentration, 0.1 mg mL⁻¹). To stop the reaction, EDTA was added to a final concentration of 50 mm. A total of 10 μg of chlorophyll was loaded in each lane.