Las 4000 camera

The isolated mitochondria were heat treated at 95 °C for 7 min using the SDS-PAGE loading buffer (LPS solution, Daejeon, Republic of Korea). After separating the proteins by size using a 12% SDS-PAGE gel, they were transferred to PVDF membranes. The membranes were treated with 3% BSA for 1 h, in order to block non-specific interactions, and then incubated overnight at 4 °C with each of the following primary antibodies: anti-AIF (sc-13116), anti-cytochrome C (sc-13156), anti-PCNA (sc-56) (all from Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), anti-COX IV (#4844s), and anti-GAPDH (#2118s) (all from Cell Signaling Technology, Beverly, MA, USA). After washing with TBST, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies, goat anti-mouse IgG (1:1000; Santa Cruz Biotechnology; sc-516102-CM), and goat anti-rabbit IgG (Santa Cruz; sc-2357). The expression of the target proteins was visualized using an enhanced chemiluminescence system (ECL component from Pierce Clarity and Western ECL Substrate; Bio-Rad Laboratories, Hercules, CA, USA) and LAS-4000 camera (Fuji Photo Film, Tokyo, Japan).

Proteins were separated from isolated thylakoid membranes. First, cells were pelleted, washed, and resuspended in buffer B (25 mM MES/NaOH, pH 6.5, 10 mM CaCl₂, 10 mM MgCl₂, and 25% (v/v) glycerol). Then, they were broken using balotina/zirconia beads and Mini-Beadbeater-24 (BioSpec Products, Bartlesville, Oklahoma, USA) for 5 cycles of 30 s of breaking and 1 min in ice. Thylakoid membrane fraction was separated by centrifugation at 20,000 g for 30 min at 4 °C. The fraction containing thylakoid membrane proteins (Chl content 5 μg) was loaded in the clear-native polyacrylamide gel electrophoresis (CN-PAGE). Protein complexes from the membrane were solubilized with 10% dodecyl-β-D-maltoside (DDM) in water to obtain the sample volume/DDM = 10 v/w). Native protein complexes were separated on 4%–14% gradient polyacrylamide gel (acrylamide to bis-acrylamide ratio was 60:1) according to Wittig et al. [55 (link)]. Native gels were color-scanned and chlorophyll a fluorescence was obtained by LAS 4000 camera (Fuji, Boston, MA, USA). The color gel pictures were analyzed by the ImageJ software (FIJI distribution). Each band corresponding either to PSI (trimer or monomer) or PSII (dimer or monomer) was taken per each time point. Areas of the peaks were analyzed and normalized to the time 0 min.