Hrp conjugated goat anti rabbit igg secondary antibody

The plant materials including Arabidopsis seedlings and tobacco leaves were ground into fine powder in liquid N₂ and added same volume of SDS loading buffer [125 mM Tris-HCl (pH 6.8), 4% (w/v) SDS, 20% (v/v) glycerol, 100 mM DTT, and 0.002% (w/v) bromophenol blue]. The extracts were thoroughly mixed by vortex and maintained on ice for 10 min. After heated at 95°C for 5 min, the mixture was centrifuged for 5 min at 16,000 g. The supernatant was treated with or without Endo Hf (New England Biolabs) treatment for 1.5 h at 37°C. Samples were then separated by 10 or 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The total proteins from the supernatants were separated by 10% SDS-PAGE. The immunoblot analyses were performed with primary antibodies, including anti-BRI1 (Mora-Garcia et al., 2004 (link)), anti-RSW2 (Liu et al., 2018 (link)), and HRP-conjugated goat anti-rabbit IgG secondary antibody (Promega). For PRX32-HA or PRX34-HA detection, immunoblot analyses were performed with peroxidase-conjugated anti-HA antibodies (Sigma). The protein signals were detected with enhanced chemiluminescence Immobilon Western HRP Substrate (Millipore) or SuperSignal West Pico PLUS substrate kit (Thermo) by a CCD imager (Tanon).

PVDF membranes were blocked in Tris-buffered saline and 0.1% Tween-20 (TBST) with 3% BSA and 5% skim milk overnight at 4 °C. Primary antibodies used in western blotting were prepared in a solution of TBST with 5% BSA at a working concentration of 1 : 1000 unless recommended otherwise by the manufacturer and included antibodies against Bid (2002), BiP (3177), Caspase-3 (9665), Caspase-8 (4790), Caspase-9 (9502), COX IV-HRP Conjugate (5247), Death Receptor 5 (8074), anti-MEK1/2 (9126), anti-ERK1/2 (137F5) (4695), anti-phosph-MEK1/2 (Ser217/221) (9154) and anti-phospho-ERK1/2 (Thr202/Tyr204) (4376) all from Cell Signaling Technology (Danvers, MA, USA); Death Receptor 4 (ab8414; AbCam, Cambridge, MA, USA); Anti-β-actin (A4700; Sigma); and HRP-conjugated goat anti-rabbit IgG (sc-2020; Santa Cruz Biotechnology, Santa Cruz, CA, USA). HRP-conjugated goat anti-rabbit IgG secondary antibody (Promega, Madison, WI, USA; SA1-9510) was used at a concentration of 1 : 5000 and detected using an enhanced chemiluminescence detection system (Amersham, Pittsburgh, PA, USA; RPN2132, RPN2133). Semiquantitative densitometry was performed using NIH Image J software (ImageJ, US National Institutes of Health, Bethesda, MD, USA).