Goat anti mouse igg antibody

For detection of BZR1 protein, the samples harvested from BZR1:OE plants were ground into powder in liquid nitrogen and homogenized in extraction buffer (100 mM HEPES, pH7.5, 5 mM Na₂-EDTA, 5 mM EGTA, 10 mM Na₃VO₄, 10 mM NaF, 50 mM β-glycerophosphate, 1 mM phenylmethylsulphonyl fluoride, 10% (w/v) glycerol, 7.5% (w/v) polyvinylpolypyrrolidone (PVP), and 0.2% (w/v) β-mercaptoethanol), followed by centrifugation at 13,000× g for 20 min. To determine the concentrations of proteins, a Bio-Rad protein assay kit was used. After adjusting the concentration to the same level, proteins were then mixed with 5× loading buffer (125 mM Tris-HCl, pH6.8, 5% (w/v) SDS, 25% (v/v) glycerol, 25% (v/v) β-mercaptoethanol, 3.13 mg bromophenol blue/5 mL buffer) and heated at 95 °C for 10 min. SDS–polyacrylamide gel 12% (w/v) electrophoresis (SDS-PAGE) was performed to resolve the protein extracts. After electrophoresis, proteins were transferred to a nitrocellulose membrane and HA was identified (Pierce, 26183, Waltham, MA, USA). After incubation with a goat anti-mouse IgG antibody (Millipore, AP124P, Germany), the complexes on the blot were visualized using SuperSignalTM WEST Pico Chemiluminescent Substrate (Thermo Fisher Scientific, 34080, Waltham, MA, USA) following the manufacturer’s instructions.

ChIP was carried out using the EpiQuiK^TM Plant ChIP kit (Epigentek, P-2014, Farmingdale, NY, USA) as described in the manufacturer’s protocol. Approximately 1 g of leaf samples was collected from plants at 6-leaf stage. To analyze the binding of BZR1 to RBOH1 and CBFs, samples were collected from BZR1:OE and WT plants treated at 8 °C for 8 h. To analyze the role of RBOH1 in BZR1 binding ability, samples were collected from RBOH1-silenced or non-silenced plants with application of H₂O or 200 nM EBR for 12 h. To analyze the role of H₂O₂-dependent glutathione homeostasis in BZR1 binding ability, samples were collected from RBOH1-silenced or non-silenced plants with application of H₂O, 5 mM H₂O₂, or 5 mM GSH. The DNA fragments combined with BZR1 protein were co-immunoprecipitated with an anti-HA antibody (Pierce, 26183). Goat anti-mouse IgG antibody (Millipore, AP124P, Germany) was used as the negative control. The enriched DNA fragments were quantified by qRT-PCR using the primers listed in Table S5. The ChIP-qPCR data were analyzed according to the calculation method described by Patrik [48 (link)].

Dexamethasone (Dex) was purchased from Taiji Group (Chongqing, China). Dulbecco’s Modified Eagle Medium (DMEM) was purchased from Hyclone (Logan, UT, USA). Fetal bovine serum (FBS), L-glutamine and antibiotics, mouse anti-actin antibody, goat anti-mouse IgG antibody and goat anti-rabbit IgG antibody were purchased from Millipore (Billerica, MA, USA). Mouse anti-HO-1 antibody, mouse anti-CypD antibody, rabbit anti-phospho-p38 antibody and rabbit anti-p38 antibody were purchased from abcam (Cambridge, MA, USA). Mitosox red, MitoTracker green and Lipofectamine® RNAiMAX Transfection Reagent were purchased from Thermo Scientific (Waltham, MA, USA). ON-TARGET plus Mouse ppif siRNA (L-062722) and control siRNA (D-001810) were purchased from Dharmacon Research (CO, USA). Annexin V-FITC/propidium iodide (PI) Apoptosis Detection kit was purchased from KeyGEN BioTECH (Nanjing, China). Other reagents not mentioned here were purchased from Sigma-Aldrich (St. Louis, MO, USA).