Goat anti mouse igg1 hrp

To detect exosomal markers (Alix, CD9, and HSP-70), pEVs were lysed and proteins extracted in RIPA buffer (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1% v/v Nonidet P-40, 0.5% v/v sodium deoxycholate, and 0.1% SDS) supplemented with protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, United States). After heating for 5 min at 95°C, pEV lysates were centrifuged for 20 min at 12,000 g, and supernatant was collected. By 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), proteins were separated by electrophoresis and thereafter transferred to a 0.1-mm nitrocellulose blotting membrane (GE Healthcare, Buckinghamshire, United Kingdom). After blocking with 2% BSA in TBS-T (20 mM Tris–HCl (pH 7.4) and 0.01% Tween-20), blots were incubated with primary antibody overnight at 4°C with dilutions as mentioned in Supplementary Figure S-1. Blots were washed three times with TBS-T and subsequently incubated with HRP-tagged secondary antibody (goat-anti-mouseIgG1-HRP, Santa Cruz, 1:5,000) for 2 h at RT. Chemiluminescence reactions were carried out using the ECL™ Prime Western Blot Detection Reagent (GE Healthcare, Buckinghamshire, United Kingdom), and protein bands were visualized on a GE ImageQuant LAS-4000.

Delphinidin-3-glucoside (ANC) was purchased from Ziguang (Nanjing, China). FA-g-MD was prepared by our group. 2,2′-diphenyl-1-picrylhydrazyl radical (DPPH) and 2′,7′-dichlorofluorescin diacetate (DCF-DA) were purchased from Aladdin Reagent (Shanghai, China). HT-29 cells were obtained from Chinese Academy of Sciences (Kunming, China). DMEM, DMEM/Nutrient Mix F12 (1:1) medium, fetal bovine serum, penicillin/streptomycin and molecular protein marker were sourced from Invitrogen GmbH (Karlsruhe, Germany). Cell culture consumable materials were purchased from Greiner Bio-One (Essen, Germany). Monoclonal antibody quinone oxidoreductase 1(NQO1), glutathione reductase (GSR), γ-glutamatecysteine ligase catalytic submit (γ-GCLC), and β-actin mouse monoclonal (IgG1) and secondary antibodies (goat anti-mouse IgG1-HRP, goat anti-rabbit IgG-HRP) were purchased from Santa Cruz Biotechnology (Heidelberg, Germany). All organic solvents and other chemicals were of analytical grade and complied with the standards needed for cell culture experiments.

Protein conjugates and controls were analyzed by Western blot. Cell lysates were loaded into NuPage 12% or 4–12% Bis-Tris gel wells (ThermoFisher Sci.) for gel electrophoresis, then transferred to nitrocellulose membrane using iBlot Transfer Stacks and the iBlot Gel Transfer Device (ThermoFisher Sci.). Blots were initially stained with Ponceau stain, then blocked 5% skim milk and probed with anti-Fentanyl (Cal BioReagents) and goat anti-mouse IgG1-HRP (Santa Cruz). After imaging, blots were stripped with Restore Plus Stripping Buffer (ThermoFisher Sci.) then re-developed with mouse anti-CRM antibody (Antibody and Immunoassay Consultants) and goat anti-mouse HRP antibody (Santa Cruz). Blots were imaged with Pierce™ ECL Western Blotting Substrate (ThermoFisher Sci.) and Amersham Imager 600.