As06 172 100

TAP-dark grown cells were pelleted by centrifugation, resuspended in an extraction buffer containing 5 mM HEPES-KOH, pH 7.5, 100 mM dithiothreitol, 100 mM Na₂CO₃, 2% (w/v) SDS, and 12% (w/v) sucrose, and lysed by boiling for 1 min. Extracted proteins were separated on SDS-PAGE (12% precast polyacrylamide gels, Bio-Rad) using tubulin as a loading and normalization control. Polypeptides were transferred onto polyvinylidene difluoride membranes using a semidry blotting apparatus (Bio-Rad) at 15 volts for 30 minutes. For western blot analyses, membranes were blocked for 1 h at room temperature in Tris-buffered saline-0.1% (v/v) Tween containing 5% powdered milk followed by a 1 h incubation of the membranes at room temperature with the primary antibodies in Tris-buffered saline-0.1% (v/v) Tween containing powdered milk (3% [w/v]). Primary antibodies were diluted according to the manufacturer’s recommendations. All antibodies were from Agrisera and the catalog numbers for the antibodies against CP43, PsaA, ATPC, and α-tubulin were AS11–1787, AS06–172-100, AS08–312, and AS10–680, respectively. Proteins were detected by enhanced chemiluminescence (K-12045-D20, Advansta) and imaged on a medical film processor (Konica) as previously described^{9 (link)}.