Rat α gfp

One hundred mg of N. benthamiana leaves transiently expressing relevant proteins were ground to a fine powder with liquid nitrogen and 2.5 volumes of extraction buffer (50 mM Tris-HCl, pH 7.5, 2 mM MgCl₂, 150 mM NaCl, 1% triton x-100, 140 mM β-mercaptoethanol, 2 mM PMSF and 1 mM EDTA-free protease inhibitor cocktail) were added. Samples were incubated in a rotating wheel at 4°C for 20 min before centrifugation. Supernatant samples were collected and boiled after adding sample buffer [8% SDS, 40% glycerol, 200 mM Tris-Cl, pH 6.8, 388 mM dithiothreitol (DTT), and 0.1 mg/ml bromophenol blue dye]. Samples were run in SDS-PAGE and blotted onto nitrocellulose membranes. The following primary antibodies were used: mouse α-mCherry (Chromotek), rat α-GFP (Chromotek) and rabbit α-Luciferase (Sigma–Aldrich).

Co-immunoprecipitation assays were performed as described by Leibman-Markus [47 (link)]. N. benthamiana leaves transiently co-expressing LeEIX2-HA and SlDRP1B-GFP were harvested 40 h after infiltration. Leaf petioles were immersed in EIX 3 μg/mL (or water as mock) for seven minutes and then transferred to the water for an additional seven minutes. A total of 500 mg leaf tissue was used for co-immunoprecipitation, with 13 μL α-HA Affinity Matrix (Roche, Indianapolis, ID, USA). Samples were run in SDS-PAGE, blotted onto nitrocellulose membranes, and incubated with antibodies as required: rat α-GFP (Chromotek, Planegg-Martinsried, Germany) and mouse α-HA (Biolegend, San Diego, CA, USA).