Sting antibody

Cells were lysed with gentle lysis buffer (Cell Signaling) for 15 mins, and then the cell lysate was spun down at 16,000 g for 10 mins. STING antibody (Proteintech) was added into the cell lysate at 1 µg/mL and incubated overnight at 4°C with rotation. The next day, 20 µL of Pierce protein A/G magnetic beads (Thermo Fisher Scientific) were added into each sample and incubated at 4°C for 2 hr to capture STING protein. Samples were then washed with lysis buffer five times, and protein was dissociated from beads by heating at 97°C for seven mins.

Cells on glass slides were fixed by neutral paraformaldehyde (4%) for 30 minutes and permeabilized using PBS containing 0.01% Triton X‐100 for 15 minutes. Next, cells were incubated with rabbit anti‐STING (1:50) antibody or rabbit anti‐CREB (1:50) antibody at 37°C for 2 hours, followed by incubation with the appropriate secondary antibody (Jackson, USA) for 1 hour at 37℃. STING antibody was from ProteinTech (USA) and CREB antibody was purchased from Santa Cruz (USA). Additionally, cells were incubated with 4′,6‐diamidino‐2‐phenylindole (DAPI) as a nuclear counterstain. A confocal laser scanning microscope (Olympus BX43, Japan) was used for confocal microscopy.