Irf4 antibody

A peroxidase-labeled mouse anti-human IgE Fc antibody and peroxidase-labeled goat anti-mouse IgE, IgG1 and IgG2a Fc antibodies were obtained from SouthernBiotech, USA (9160-05, 1110-05, 1070-05 and 1155-05); tetramethylbenzidine (TMB) was purchased from Solarbio, China (R1200); aluminum hydroxide was obtained from Thermo Fisher, USA (77161); and LPS was purchased from Sigma, USA (L3012). ELISA kits for IL-4, IFN-γ and TNF-α were obtained from Ebioscience, USA (88-7044, 88–7314 and 88–7324); ELISA kits for IL-5 and IL-13 were purchased from 4 A Biotech, China (CME0003, CME0009); an IRF4 antibody was obtained from Cell Signaling, USA (4964); an antibody against GAPDH was procured from Proteintech, China (10494-1-AP); a TLR4 signaling inhibitor was purchased from Invivogen, USA (CLI-095); an anti-mouse TLR2 Ab was obtained from Biolegend, USA (121802); the PE-CD80, PE-CD83, FITC-MHCII and FITC-CD40 antibodies were obtained from Ebioscience, USA (12–0801, 12–0831, 11–5321 and 11–0402); and mouse GM-CSF and IL-4 were purchased from Sino Biological, China (51048-M07H, 51084-M08B). Anti-CD3 and anti-CD28 antibodies were obtained from Ebioscience, USA (16-0031-82, 16-0281-82).

Two cell lines defined as lethal by CRISPR screening (RVH421 and WM1799) and two non‐lethal (WM983B and HT144) melanoma cell lines were transfected with siRNAs. These siRNAs were ON‐TARGETplus SMARTpools (Dharmacon, Lafayette, CO, USA) designed against interferon regulatory factor 4 (IRF4), ERH (positive control/essential gene) or with a non‐targeting pool (negative control) according to the manufacturer's instructions. Cells were retransfected after 3 or 6 days and harvested for analysis after 10 days. Cells were stained with Annexin V‐PE (Biolegend, San Diego, CA, USA) and DAPI (Sigma‐Aldrich, St. Louis, MO, USA) and analysed by flow cytometry using a BD Fortessa II and FlowJo version 10 (Becton Dickinson, Franklin Lakes, NJ, USA). Successful gene knock‐down was confirmed by western blot analysis using an IRF4 antibody (Cell Signaling Technology, Danvers, MA, USA). GAPDH (Cell Signaling Technology, clone 14C10) or vinculin (Sigma‐Aldrich, clone V284) were used as loading controls. Cells were also analysed for c‐MYC protein expression using antibody clone Y69 (Abcam, Cambridge, UK).