Blue-native gel electrophoresis

The status of the PEP complex assembly was analyzed via blue-native polyacrylamide gel electrophoresis (BN-PAGE) and immunoblot analyses. Seedlings (100 mg) were harvested and flash-frozen in liquid nitrogen, ground to a fine powder, and resuspended in three volumes (mg/µL) of BN lysis buffer containing 100 mM Tris-Cl pH 7.5, 10 mM MgCl₂, 25% glycerol, 1% Triton X-100, 10 mM NaF, 5 mM β-mercaptoethanol, and 1× EDTA-free protease inhibitor cocktail (MilliporeSigma). Protein extracts were mixed with the BN sample buffer containing 1× NativePAGE sample buffer, 50 mM 6-aminocaproic acid, 1% n-dodecyl β-D-maltoside (DDM), and Benzonase nuclease using a NativePAGE Sample Prep Kit (Thermo Fisher Scientific). After incubation for 1 h at room temperature, BN-PAGE protein samples were mixed with 0.25% NativePAGE Coomassie blue G-250 sample additive and centrifuged at 17,500 × g for 10 min at 4 °C. Protein samples in the supernatant were separated on 4–16% NativePAGE Bis-Tris protein gel at a constant 30–40 V for 3 h at 4 °C with Dark Blue Cathode Buffer until the blue dye migrated through one-third of the gel and further separated at a constant 20–25 V overnight at 4 °C with Light Blue Cathode Buffer. The separated proteins were transferred onto a polyvinylidene difluoride membrane at a constant 70 V for 7 h at 4 °C. The membrane was destained with methanol for 3 min, probed with the indicated primary antibodies, and incubated with the indicated secondary antibodies mentioned above.