BN-PAGE

Constructs were transformed into Agrobacterium tumefaciens strain GV3101/pMP90 and used for transient expression in Nicotiana benthamiana. A. tumefaciens cells containing NRG1.1 WT and variants constructs were grown in LB media at 28°C. Antibiotic concentrations used (in μg/mL) Kanamycin 100, Gentamycin 50 and Spectinomycin 100, Rifampicin 100. The overnight cultures were centrifuged for 8 min at 8,500 rpm and the pellets were resuspended in induction buffer (10 mM MgCl2, 10 mM MES pH 5.6, 150 μM acetosyringone). OD₆₀₀ was adjusted to 0.1 (35S::P19) and 0.8 for NRG1.1 and ADR1 constructs. 

Agrobacteria mixtures were infiltrated into young leaves of 4-6 weeks old N. benthamiana WT plants using a 1-ml needleless syringe. The N. benthamiana plants were grown on soil under 12h light / 12h dark cycles (24°C/22°C, 65% humidity). Induction was done 24 hours post infiltration using 100 μM β-estradiol (Sigma-Aldrich; St. Louis, USA) and 0.001% [v/v] Silwet L-77 by spraying. Tissues for protein assays were collected 6 h post induction. 

For blue native polyacrylamide gel electrophoresis, modified GTEN buffer (60) [10% glycerol, 100 mM Tris-Cl pH7.5, 1mM EDTA, 150mM NaCl, 5mM DTT, 0.5% n-dodecyl-B-D-maltoside (DDM) and 1x Sigma plant protease inhibitor cocktail] was added to the homogenized tissue at a ratio of 5 uL per mg (FW) tissue. Lysate was cleared by centrifuge at 20,000 x g for 30 min at 4°C. The supernatant was transferred to a new tube and mixed with 4x NativePAGE Sample buffer (Invitrogen). NativePAGE^TM 5% G-250 Sample Additive were added to eac tube at 1/4th  of the detergent concentration. Proteins were resolved in 3-12% and NativePAGE^TM (Invitrogen) gels then transferred to PVDF membrane using iBlot^TM 2 gel transfer device according to the instructions from the manufacturer.

After the transfer the membranes were destained twice with methanol for 30 seconds and rinsed with deionized water. Membranes were blocked with 5% non-fat milk TBST and probed with primary anti-GFP (Roche, Cat. #: 11814460001, 1:1000) and secondary HRP-conjugated anti-mouse antibody (R&D Systems, Cat. #: HAF007, 1:5000).