4 to 20 mini proteantgx precast gels

Leaf tissue of N. benthamiana collected from biological replicates (plants) of the same treatment was used for protein extraction. Frozen leaf tissue was lysed using TissueLyser II (Qiagen, Germantown, MD, U.S.A.) and suspended in dithiothreitol (DTT)-containing lysis buffer (50 mM HEPES-KOH, pH 7.4, 110 mM KOAc, 2 mM MgCl 2 , 0.4% TritonX-100, 2.5 mM DTT, and 1× protease inhibitor cocktail from Invitrogen) at a 1:5 ratio (wt/vol) (DeBlasio et al. 2015; (link)Osterbaan et al. 2021) (link). The protein supernatants were mixed at a 1:1 ratio (vol/vol) with loading buffer (2× Laemmli sample buffer [Bio-Rad] with 5% β-mercaptoethanol), and were incubated at 95°C for 5 min before being loaded onto 4 to 20% Mini-PROTEAN TGX precast gels (Bio-Rad). Loading buffer was used as a loading blank, and the Precision Plus Protein Dual Xtra Standards (Bio-Rad) were used as a protein ladder. Proteins were separated by electrophoresis on a Mini-PROTEAN Tetra Cell (Bio-Rad) and were then wet-transferred to 0.45 μm nitrocellulose membranes (Bio-Rad) at 4°C. An anti-EGFP mouse monoclonal antibody (F56-6A1.2.3 from Invitrogen) was used as a primary antibody at a 1:1,000 dilution, and alkaline phosphatase-conjugated goat anti-mouse immunoglobulin G (H+L) (Invitrogen) was used as a secondary antibody, at a 1:5,000 dilution with nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate substrates.