Protein Phosphorylation (Western Blot)

Protein expression and phosphorylation (for IRF3 and TBK1) was assessed in transiently transfected HEK 293 T cells and lentivirus-transduced stable BJAB cell lines, expressing the STING alleles of interest. For protein expression and western blotting experiments, cells were incubated overnight in antibiotic-free medium and then stimulated with 2’3’cGAMP (Invivogen; 20 µg/mL for the HEK 293 T cells and 100 µg/mL for the BJAB cells) for different timepoints (15 min, 30 min, 1 h, 2 h, and 4 h). The cells were lysed in RIPA lysis buffer (Sigma) containing protease and phosphatase inhibitors (protease inhibitors cOmplete™ and phosphatase inhibitors PhosSTOP™, Roche). Lysates were centrifuged at 16,000 RPM and 4°C for 20 min. Protein concentrations were quantified using the Pierce BCA Protein Assay Kit (ThermoFisher Scientific; Minneapolis, MN), and equal protein amounts (2 to 3µg) were used for western blot analysis. Laemmli buffer (Bio-Rad; Hercules, CA) with β-mercaptoethanol was added to the samples, and the lysates were denatured by incubating at 95°C for 5 min and were centrifuged at 16,000 RPM for 1 min. Samples were loaded onto 4–20% Criterion™ gels (Bio-Rad; Hercules, CA), and then proteins were transferred to Trans-Blot^® Turbo Midi PVDF membranes (Bio-Rad; Hercules, CA) using the Trans-Blot^® Turbo™ Transfer System (Bio-Rad; Hercules, CA). Blots were blocked with 3% BSA and probed overnight (at 4°C) with primary monoclonal rabbit anti-STING (cat. # 13647), anti-TANK-binding kinase 1 (TBK1, cat. # 3504), anti-pTBK1 (cat. # 5483), anti-interferon regulatory factor 3 (IRF3, cat. # 4302), and anti-pIRF3 antibody (cat. # 4947) (all from Cell Signaling Technologies; Beverly, MA), or mouse monoclonal anti-alpha tubulin antibody (cat. # 40742, Abcam; Cambridge, MA) for loading control. Membranes were washed and incubated for 1 h at room temperature with the appropriate HRP-labeled pre-absorbed goat anti-rabbit (cat. # sc-2054) or anti-mouse (cat. # sc-2055) secondary antibodies (Santa Cruz Biotechnology, Inc.; Dallas, TX). The membranes were washed, developed using Clarity Western ECL Substrate Solution (Bio-Rad; Hercules, CA) for 10 min, and imaged using the ChemiDoc™ Touch Gel Imaging System (Bio-Rad; Hercules, CA). Comparisons were assessed using Student’s t-test.