Instab protease cocktail

The DF-1 cells were plated in 12-well plates at 1×10⁶/mL and then transfected with a total of empty plasmid or GoSTING expression plasmid. At thirty-six hours post-transfection, cells were washed twice with phosphate buffer saline (PBS) (Gibco) and then lysed with a cell lysis buffer (Beyotime, Shanghai, China) containing an InStab™ protease cocktail (Yeasen, Shanghai, China) and phenylmethylsulfonyl fluoride (PMSF) (Yeasen). Lysates were centrifuged at 13,000 rpm for 15 minutes to obtain the supernatant and were eluted with a 5×SDS-PAGE loading buffer (Yeasen) and boiled for 10 min. Then the cell lysates were separated via SDS-PAGE and analyzed by Western blotting. Images were collected with the Tanon 5200 imaging system (Tanon, Shanghai, China), as described in our previous study (34 (link)).

The DF-1 cells were plated in 12-well plates at 1 × 10⁶/1 mL and then transfected with empty plasmids or various expression plasmids. After 24 h, the transfected cells were washed twice with phosphate buffer saline (PBS) (Gibco) and then lysed with a cell lysis buffer (Beyotime, Shanghai, China) containing an InStab™ protease cocktail (Yeasen, Shanghai, China) and phenylmethylsulfonyl fluoride (PMSF) (Yeasen). The cell lysates were centrifuged at 13 000 rpm for 15 min to obtain the supernatant, and a 5 × SDS-PAGE protein loading buffer (Yeasen) was added. The lysates were then boiled for 10 min. The proteins isolated from the cell lysates were separated via SDS-PAGE and analyzed using a Western blot. Images were obtained using the Tanon 5200 imaging system (Tanon, Shanghai, China), as described in our previous study [42 (link)].