Phospho sting

C32 or SK-MEL-28 cells were lysed in radioimmunoprecipitation assay (RIPA) buffer supplemented with protease and phosphatase inhibitors. Protein concentrations were estimated using Pierce BCA Protein assay kit (Thermo Fisher Scientific), as per the instructions. Equal amounts of proteins were separated on 4–20% Mini-PROTEAN TGX Stain-Free Gels (#4568094, Bio-rad). Proteins were then transferred onto PVDF membranes and probed with the following antibodies: STING/TMEM173 (NBP2-24683, Novus), phospho-STING (#19781, Cell Signaling technology), TBK1 (#3504, Cell Signaling Technology), phospho-TBK1 (#5483, Cell Signaling Technology), NRF2 (ab137550, Abcam). Beta actin or Calnexin were used as loading controls. After incubation with secondary horseradish peroxidase (HRP)-conjugated antibodies, the blots were washed and developed using enhanced chemiluminescence reagent and imaged in the ImageQuant LAS4000.

After incubation cells or tissue were lysed in sample buffer containing 50 mmol/L Tris-HCl pH 6.8, 2% w/v sodium dodecyl sulfate, 10% glycerol, 0.0025% w/v bromophenol blue, and 5% β-mercaptoethanol. Protein samples were then loaded and resolved on Mini-PROTEAN TGX Gels and then transferred onto polyvinylidene fluoride membranes (BioRad) using a Trans-Blot Turbo Transfer System (BioRad). The polyvinylidene fluoride membranes were then blocked with 10% nonfat dry milk or BSA in PBS with 0.1% v/v Tween (PBS-T) for 1 hour, and then incubated with the primary antibodies. Membranes were developed with the Pierce ECL detection system (Thermo-Fisher Scientific) and signal detected using photographic film (GE Healthcare, catalog no. 28-9068-37) and an automatic processor (Fuji RG II). Samples were immunoblotted for Phospho-VASP (Ser239; Cell Signaling, No. 3114), Phospho-VASP (Ser157; Cell Signaling, no. 84519), cGAS (Invitrogen, no. PA5-76367), Phospho-TBK1 (Ser172; Cell Signaling, no. 5483), TBK1 (Cell Signaling, no. 3013), Phospho-STING (Ser365; Cell Signaling, no. 72971), CD31 (Abcam, no. ab28364), MRP1 (Santa Cruz, no. sc-18835), LRRC8A (Santa Cruz, no. sc-517113), GAPDH (Cell Signaling, no. 2118), and β-actin (Sigma-Aldrich, no. A5316).

Total protein lysates from cells were prepared using sonication with PRO-PREP™ Protein Extraction Solution (iNtRON Biotechnology, Seongnam, South Korea). Protein levels were determined using a Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Equivalent amounts of proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to membranes. The membranes were subsequently incubated with primary antibodies for 24 h at 4 °C. Primary antibodies: anti-VP3, β-actin (Santa Cruz Biotechnology, Dallas, TX, USA), STING, Phospho-STING, TBK1/NAK, Phospho-TBK1/NAK, IRF3, Phospho-IRF3 (Cell Signaling, Denver, MA, USA). Thereafter, secondary Abs, goat anti-mouse IgG F(ab′)2, polyclonal Abs (HRP conjugated) (Enzo Life Sciences, Farmingdale, NY, USA), and anti-rabbit IgG (HRP-linked antibody) (Cell Signaling, Denver, MA, USA), were added for 2 h at 20 °C. Proteins were detected using West Femto Maximum Sensitivity Substrate (Abbkine, Atlanta, GA, USA) and visualized using an ImageQuant LAS 4000 Mini System (Cytiva, Marlborough, MA, USA). Chemiluminescence intensity was analyzed using ImageJ software (NIH, Bethesda, MD, USA).