Albumin bovine 5 bsa 5

Total protein was extracted using 2% SDS lysis buffer including protein phosphatase inhibitor (Applygen, Beijing) and heated for 10 min. Protein samples were separated by 8% (v/v) SDS-PAGE gels and transferred onto nitrocellulose membranes (Millipore, Ireland). After blocking in 5% (w/v) Albumin Bovine-V (BSA-V, Solarbio, China) for 1 h at room temperature, the protein bands were incubated with primary antibodies at a dilution ratio of 1:1000 overnight at 4 °C. The primary antibodies contain anti-GAPDH (FL-335; Santa Cruz Bio technology), anti-TGM2 (Proteintech), anti-PD-L1 (Proteintech), anti-STAT3 (CST) and anti-Phospho-STAT3 (Ser705, CST), anti-Akt (CST), and anti-Phospho-Akt (Ser473, CST), anti-P65 (Abcam) and anti-Phospho-P65 (Ser536, Abcam). The protein bands were incubated in HRP-conjugated secondary antibodies (Zsbio, China, 1:5000) at room temperature for 1 h.

We performed western blotting according to a standard protocol. Total cell lysates were extracted using 2% SDS lysis buffer with a protein phosphatase inhibitor mix (Applygen, Beijing), and 30 μg of total protein was prepared for electrophoresis through 8% or 12% (v/v) SDS–PAGE gels. After electrophoresis, the separated protein bands were transferred onto nitrocellulose membranes (Millipore, Ireland) and were blocked in 5% (w/v) Albumin Bovine-V (BSA-V, Solarbio, Beijing) for 1.5 h at room temperature. The primary antibodies used were as follows: anti-GAPDH (FL-335; Santa Cruz Biotechnology), anti-CD59 (HPA026494, Sigma-Aldrich), anti-CD163 (ab182422, Abcam), anti-arginase 1(Arg-1) (16001-1-AP, Proteintech), anti-IFN-γ (ab9657, Abcam), anti-iNOS (ab3523, Abcam), anti-IL-6 (ab6672, Abcam), anti-STAT3 (D1B2J, Cell Signaling Technology), and anti-phospho-Stat3 (Ser727, Cell Signaling Technology). The quality of loading and transferring was assessed by immunostaining with the anti-GAPDH antibody. The membranes were incubated with the primary antibodies at a dilution ratio of 1:1000 overnight at 4 °C. After washing three times in TBST, the membranes were incubated in 1:5000 HRP-conjugated secondary antibodies (Zsbio, Beijing) at room temperature for 1 h. Finally, the membranes were washed three times in TBST and visualized using an ECL Kit (Applygen, Beijing).