Pvdf transfer membrane

The concentration of cell extracts was measured by a Bradford Protein Assay Kit (Beyotime). An equal amount of total protein extracts was separated by 12% SDS-PAGE and transferred to PVDF transfer membranes (Bio-Rad Laboratories, Hercules, CA, USA) and immunoblotted with various antibodies. The signals were detected by incubation with a SuperSignal West Pico Chemiluminescent detection system (Pierce Chemical Co, Rockford, IL, USA). Images were generated using a ChemiDoc XRS (Bio-Rad Laboratories), and protein band intensity was measured using Quantity One software. Three independent experiments were performed.

Lewis lung carcinoma cells and G‐MDSC were lysed in radioimmunoprecipitation (RIPA) buffer (Auragene Bioscience, Hunan, China). The extracted protein samples were separated by 10% or 12% SDS‐PAGE and transferred onto polyvinylidene fluoride (PVDF) transfer membranes (Bio‐Rad, Hercules, CA, USA). After blocking with 5% (w/v) skimmed milk for 2 hours on a shaker at room temperature, membranes were incubated overnight at 4°C with primary antibodies specific for HMGB1, Arginase‐1 (Arg‐1), total caspase 3, cleaved caspase‐3, total stat 3 (rabbit; CST, Danvers, MA, USA), Bax, Bcl‐2 (Santa Cruz Biotechnology, Shanghai, China) and β‐actin (Abcam, Shanghai, China). Then, membranes were washed with TBS supplemented 5% Tween 20 (TBST) (for 20 minutes, 3 times) and subjected to secondary HRP‐labeled goat anti‐rabbit/mouse antibodies (Abcam Cambridge, UK). Detection was carried out with Luminescent Image Analyzer (Image Quant LAS4000mini, GE Healthcare, China) and relevant blots were quantified by densitometry using the Gel‐Pro 32 software.