Rat anti gfp

Co-immunoprecipitation assays were performed as described by Leibman-Markus et al. (2017) (link): N. benthamiana leaves transiently coexpressing LeEIX2-GFP or LeEIX2 and SlDRP2A-HA were harvested 40 h after infiltration. Leaf petioles were immersed in EIX 300 μg/ml (or water as mock) for 7 min, and then transferred to water for an additional 7 min. Five hundred mg leaf tissue was used for co-immunoprecipitation, with 13 μl GFP-TrapA beads (Chromotek, Planegg-Martinsried, Germany). Beads were incubated for 4 h and then washed five times with detergent free EB. Immune-precipitated (IP) and input samples were run in SDS-PAGE, blotted onto nitrocellulose membranes and incubated with antibodies as required: rat anti-GFP (Chromotek, Planegg-Martinsried, Germany) and mouse anti-HA (BioLegend, CA, United States).

Cell lysates from third instar wing imaginal discs and brains were prepared in lysis buffer (50 mM Tris-HCl (pH 7.5), 300 mM NaCl, 0.1 mM EDTA, 1% Triton X-100, 0.1% SDS, 5% Glycerol, 1 mM PMSF, 1/10 tablet of Complete Mini Protease Inhibitor Cocktail) on ice. Protein samples were loaded onto a 10% polyacrylamide gel, along with Chameleon Duo Pre-stained Protein Ladder (LI-COR, P/N 928–60000) as a molecular weight ladder and run at 150 V. After SDS-PAGE, proteins were transferred onto a nitrocellulose membrane (Bio-Rad, 162–0115, 0.45 μm) in transfer buffer (25 mM Tris, 192 mM glycine, 20% (v/v) methanol) using the wet-tank method with a current density of 300 mA for 1 h. Prior to antibody incubation, membranes were blocked with 5% milk in PBS. Membrane was incubated with primary antibodies in PBS-T (1% Triton-X in 1xPBS)—rat anti-GFP (Chromotek, 3H9-100, 1:1,000) and mouse anti-α-tubulin (Sigma, T9026, 1:5,000) on a rotative plate at 4°C overnight. Primary antibodies were removed and washed in dH₂0 twice for 5 min each. Membrane was then incubated with secondary antibodies diluted in PBS-T—donkey α-mouse secondary (LI-COR, 926–68072, 1:20,000) labelled with a 700-nm IRDye and goat α-rat (LI-COR, 926–32219, 1:20,000) labelled with an 800-nm IRDye. Membrane was washed in dH₂0 twice for 5 min each before detection using the Image Studio Software on the Odyssey SA system (LI-COR).

Western blot analysis was conducted as described previously (26 (link)). Primary antibodies diluted in 5% milk/PBS-T were: mouse anti-actin (1:2000; Merck, Germany, Cat. No. MAB1501), rat anti-MYC (1:1000; Chromotek, Germany, Cat. No. 9e1-20), rat anti-GFP (1:1000; Chromotek, Germany, Cat. No. 3h9-20) and rabbit anti-REGE-1 polyclonal antibody raised against the first 119 amino acids (1:1000; SDIX, USA). Detection was performed with horseradish peroxidase-conjugated secondary antibodies: horse anti-mouse (1:5000; Cell Signaling, USA, Cat. No. 7076S), goat anti-rat (1:5000; Cell Signaling, USA, Cat. No. 7077S) and goat anti-rabbit (1:5000; Cell Signaling, USA, Cat. No. 7074S), radiance ECL detection reagent (Azure Biosystems, Germany, Cat. No. AC2204) and the c600 imaging system (Azure Biosystems, Germany). Western blotting was performed for all the biological replicates. One representative blot is shown in the results.