PD-L1 palmitoylation

Evaluation of FASN-regulated PD-L1 palmitoylation was performed using the commercially available CAPTUREome™ S-palmitoylated protein kit (Cat. #K010-311, Badrilla, UK) according to the manufacturer’s instructions. The assay is based on the acyl resin-assisted capture (RAC) methods and facilitates the determination of post-translational protein modification with a palmitate group using four steps, namely free thiol blockade, thioester bond cleavage, nascent thiol capture on Sepharose, and analysis [78]. Briefly, equal amounts of protein (1–2 mg) were added to 500 μL of blocking buffer (buffer A with thiol blocking reagent) and shaken at 40 ^oC for 4 h. Proteins were precipitated at −20 ^oC for 20 min with the addition of three volumes of cold acetone. After centrifugation of the solution at 16,000 g for 5 min, the pellet was thoroughly washed five times with 70% acetone and completely air dried after the last wash. The pellet was redissolved in 300 μL binding buffer and incubated for 1 h at 40 ^oC in a shaking heat block. The homogenates were centrifuged at 16,000 g for 5 min to remove insoluble debris, and approximately 20 μL of each supernatant was saved as the “total input”. The pre-washed capture resin slurry (50 μL) was added to the remaining lysates, followed by the addition of 19 μL of thioester cleavage reagent. The binding reactions were performed on a rotator at room temperature for 2.5 h. The resins were washed a minimum of five times with the binding buffer. The supernatants were removed and mixed with 2×Laemmli loading buffer, heated to 60 ^oC for 10 min, and resolved by SDS-PAGE.