Anti ha horseradish peroxidase

Total proteins were extracted from leaf tissue by homogenization in extraction buffer (100 mM Tris-HCl [pH 7.5], 150 mM NaCl, 5 mM EDTA, 5% glycerol, 10 mM DTT, 2% PVPP, 1 mM PMSF, protease inhibitors cocktail [Roche], and 0.5% Triton X-100). In all experiments protein samples were equilibrated to equivalent concentrations of total proteins. Extracted proteins were fractioned by 8%–10% SDS-PAGE, transferred onto HybondTM-P membranes (Amersham) and incubated with anti-HA-horseradish peroxidase (Roche) or anti-GFP-horseradish peroxidase antibody (Milteny Biotec). Immunodetection was performed with ECL chemiluminiscence reagent (GE Healthcare) or Supersignal West Femto (Thermo Scientific).

The levels of NR and HA-tagged proteins were analyzed in total protein extracts by SDS–PAGE, blotting onto nitrocellulose membranes and further probing with polyclonal anti-NR (1:1,000 dilution; Agrisera, Sweden) and anti-HA-Horse radish peroxidase (1:1,000 dilution; Roche, Switzerland) antibodies. Loading control was assessed by staining nitrocellulose membranes after blotting with Ponceau S. NR activity assays were performed as reported previously (Park et al., 2011 ) with slight modifications (Costa-Broseta et al., 2021 ). Assays included 20 µg of protein extracts in a 250-µL total volume and were performed at 25°C for 30 min.

MBP-JAZ fusion proteins were generated as previously described [21] (link). Ten-day-old Arabidopsis wild-type Col-0 seedlings and lines expressing DEX:hopX1-HA or DEX:hopX1^C179A-HA were induced with 30 µM DEX plus 0.01% Silwet L-77 or a mock solution for 24 hours. Seedlings were ground in liquid nitrogen and homogenized in extraction buffer containing 50 mM Tris-HCl, (pH 7.4), 150 mM NaCl, 10% glycerol, 0.2% NP-40, 1 mM DTT, 1 mM phenylmethylsulphonyl fluoride, 5 mM MgC_l2, 50 mM MG132 (Sigma-Aldrich), and complete protease inhibitor (Roche). After centrifugation (16,000 g at 4°C), the supernatant was collected. For in vivo PD experiments, 6 µg of resin-bound MBP fusion protein was added to 250 µg of pre-equilibrated total protein extract and incubated for 1 h at 4°C with rotation. After washing, samples were denaturalized, loaded on 8% SDS-PAGE gels, transferred to nitrocellulose membranes, and incubated with anti-HA-horseradish peroxidase (Roche). A 5 µl aliquot of MBP-fused protein of each sample was run into SDS-PAGE gels and stained with Coomassie brilliant blue to confirm equal protein loading.