Supersignal west pico plus substrate kit

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).

CdCl₂ was obtained from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China), with a purity of >98.0%. Alanine/aspartate aminotransferase (ALT/AST) kits were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). T-PER tissue extraction reagent, NE-PER nuclear and cytoplasmic extraction reagents, SuperSignal West Pico PLUS Substrate kit, and Pierce BCA Protein Assay Kit were obtained from ThermoFisher Scientific (Waltham, MA, USA). Murine IL-1β and IL-6 ELISA kits were purchased from Neobioscience (Shenzhen, China). The antibodies that were used for immunoblotting anti-Keap1, -p38, -phospho-p38, -ERK, -phospho-ERK, -JNK, -phospho-JNK, -NLRP3, -NF-κB p65, and -phospho- NF-κB p65 were purchased from Cell Signaling Technology (Danvers, MA, USA) (1:1000 dilutions). The antibodies against Nrf2 and HO-1 were all purchased from Santa Cruz (Santa Cruz, CA, USA) (all 1:200 dilutions). Antibodies against GAPDH were purchased from Abways Technology (Shanghai, China) (1:2000 dilutions). Peroxidase-conjugated goat anti-rabbit immunoglobulin IgG (H + L), anti-mouse IgG (H + L), and anti-goat IgG (H + L) were obtained from Proteintech Group (Wuhan, China). Other reagents, unless indicated, were obtained from Sigma Chemical Co. (St. Louis, MO, USA).

Tricine SDS-PAGE on a 16% acrylamide gel, followed by immunoblotting with polyclonal anti-human PRL antiserum (CL-1)^{70 (link)} or monoclonal anti PRL N-terminal antibody 5602^{15 (link)} were used for evaluation and quantitation, respectively, the presence of vasoinhibins in the conditioned medium of transduced cells. Tricine-SDS-PAGE is the preferred electrophoretic system for the resolution of proteins smaller than 30 kDa^{71 (link)}. Detection was by chemiluminescence using the peroxidase affiniPure donkey anti-rabbit or anti-mouse IgG (H&L) (Jackson ImmunoResearch, West Grove, PA) and the SuperSignal West Pico PLUS Substrate kit and the Protein simple fluorchem imager and gel documenter system (both from ThermoFisher Scientific, Waltham MA). Following the method described previously^{37 (link),72 (link)}, the vasoinhibin concentration was quantified by interpolating from a PRL standard curve (run within the same blot) the combined densitometric values of the two vasoinhibin isoforms in each conditioned media (Supplementary Fig. 7e,f). The removal of N-linked oligosaccharides with Peptide N-glycosidase F (New England Biolabs) was done according to manufacturer instructions to evaluate the presence of glycosylated vasoinhibins in the conditioned media of the transduced cells. The Quantity One 1-D Analysis Software (BioRad) evaluated optical density values.