Clarity and clarity max

Cell lysates were prepared in RIPA buffer plus proteinase inhibitors. Samples containing (20 µg) were separated by 10–15% SDS-PAGE, transferred to nitrocellulose membranes, blocked in 5% nonfat milk for 1h at RT and incubated overnight at 4 °C with the primary antibodies: MFN2 (H-68, Santa Cruz, sc-50331, dilution 1:1000, rabbit); Optineurin (C-2, Santa Cruz, sc-166576, dilution 1:1000, mouse); PINK1 (BC100-494, Novus Biologicals, dilution 1:2000, rabbit); Parkin (PRK8, ab77924, Abcam, dilution 1:2000, mouse); β-Actin (Sigma-Aldrich, A3854, dilution 1:40,000 conjugated to HRP). Secondary antibody incubation was performed for 1 h at RT using anti-mouse (m-IgGκ BP-HRP sc-516102, Santa Cruz, 1:10,000) and anti-rabbit (goat anti-rabbit IgG-HRP sc-2054, Santa Cruz, 1:10,000). Proteins were detected using ECL western blotting substrate (Pierce, Waltham, MA, USA), Clarity and Clarity Max (Bio-Rad, Hercules, CA, USA) and ChemiDoc Imaging System (Bio-Rad).

To test protein accumulation in Nb plants, three 8-mm leaf disks were harvested per sample at 2 dpi and ground in liquid nitrogen. Ground tissue was dissolved in 8-M urea buffer, vortexed for 10 min at RT, and centrifuged at 16,000g for 10 min (Ma et al., 2020) . Total protein extracts were resolved on a 10% sodium dodecyl sulfate polyacrylamide gel and subsequently transferred onto a nitrocellulose membrane using the wet transfer method. Tagged proteins in total protein or after affinity purification (see above) were detected using α-GFP antibodies (Merck; 11814460001) in a 1:5,000 dilution (1× TBST, 2% milk (w/v), 0.01% (w/v) NaAz), followed by incubation with HRP-conjugated secondary antibodies (Merck; A9044). Signal was detected by incubation of the membrane with Clarity and Clarity Max substrates (BioRad, Hercules, CA, USA; 1705061 and 1705062) using a ChemiDoc (BioRad). Membranes were stained with Ponceau S for loading control (Merck; 09276-6X1EA-F).