Anti stat1 rabbit antibody

Parental and CRISPR-edited tumour cells were seeded in a cell density of 12,500 (DU145), 15,000 (PC-3), or 30,000 (MCF7) per well in 384-well plate (Greiner Bio-One, #781080) filled with 25 µl culture media + 10% FBS. After 20–24 h incubation, the cells were treated with recombinant human IFNγ (R&D Systems, #285-IF-100) by adding an equal volume of the complete culture media containing the cytokine. After 30 min of stimulation, the culture media were removed by gentle spin using Blue Washer (BlueCatBio). Lumit immunoassay (Promega, #W1202) was performed according to manufacturer’s instrutions. The assay buffer was supplemented with Halt protease and phosphatase inhibitor cocktail (Thermo Scientific, #78443). The cells were lyzed with 12 µl per well of lysis solution (0.02% digitonin) for 20 min. The cell lystates were probed for 90 min with 12 µl of mixture of primary antibodies, which include anti-STAT1 rabbit antibody (Cell Signaling Technology, #14994), anti-pSTAT1 (Tyr701) mouse antibody (Abcam, ab29045), Lumit anti-mouse antibody-LgBiT (Promega) and Lumit anti-rabbit antibody-SmBiT (Promega). Final concentration of each antibody is 0.15 µg ml⁻¹. After 2 min incubation in the presence of the Lumit detection reagent (6 µl), the luminescence signals were detected by Infinite M1000 PRO (Tecan).

HMDMs were stimulated with BD serum or HC serum for 0, 15, 30, and 60 min. Total proteins of 1–2×10⁶ HMDMs were extracted with Minute Total Protein Extraction Kit (Invent Biotechnologies) and were quantified by BCA Assay Kit (Pierce). Proteins were loaded and electrophoresed on a 4–20% SDS-PAGE gel and were subsequently transferred to a PVDF membrane (Millipore). The membrane was blocked with tris-buffered saline-Tween 20 (TBST) containing 5% non-fat milk for 1 h at room temperature followed by incubation overnight with anti-NF-κB p65 rabbit antibody, anti-Phospho-NF-κB p65 rabbit antibody, anti-IκBα rabbit antibody, anti-JAK1 mouse antibody, anti-Phospho-JAK1 rabbit antibody, anti-STAT1 rabbit antibody, anti-Phospho-STAT1 rabbit antibody or anti-β-actin rabbit antibody (Cell Signaling Technology) at 4°C. The membrane was washed three times and incubated with HRP-conjugated secondary antibodies (EASYBIO) for 1 h at room temperature. The proteins were visualized using a ChampChemi Multiplex Fluorescence /Chemiluminescence Imager (Sage Creation Science), and the optical density data were analyzed using ImageJ software. β-actin was used as the endogenous control.