Fluorescent scanner

Adherent cells were washed with ice-cold PBS and lysed and scrapped in CelLytic^™ M buffer (Sigma) with 1X Halt^™ protease and phosphatase cocktail inhibitors (Thermo Fisher) before being centrifuged for 15 minutes at 20,000 x g at 4°C. Proteins were harvested from supernatant and quantified by using Bradford protein assay dye reagent (Bio-Rad). 25 μg of protein from whole cell extract were loaded in 4–10% acrylamide gels and transferred on nitrocellulose membranes. Membranes were stained overnight in 5% BSA-containing PBS Tween 0.1% with following antibodies: mouse anti-PLCγ (1:2000; Millipore #05–163) and rabbit anti-cGAS (mouse; 1:1000; Cell signaling #31659). Proteins were revealed with fluorescent secondary anti-rabbit (1:10000; LI-COR #926–68073) or anti-mouse antibodies (1:10000; LI-COR #926–32212) using the LI-COR fluorescent scanner.

Cells (1 × 10⁶ cells) were plated in 6 cm dishes in complete media the night before experiment. Cells were washed with PBS and placed in DMSO vehicle or compound-containing serum-free media for 2 h before being washed and collected into a lysis buffer containing protease and phosphatase inhibitors. Proteins were separated on a 4–20% Tris-Glycine precast Midi-PROTEAN TGX SDS/PAGE gel (BioRad). Proteins were then transferred to a PVDF membrane using the iBlot system (Invitrogen). Membranes were blocked in 5 % nonfat milk in TBST and incubated in primary antibodies overnight according to manufacturer instructions. Membranes were then washed in TBST and probed with secondary antibody (Li-Cor) and visualized using a fluorescent scanner (Li-Cor). Quantitation was performed using ImageJ.