Anti cd206

Cells were washed thrice with cold PBS and lysed on ice in radioimmunoprecipitation assay (RIPA) buffer supplemented with 1% Phosphatase Inhibitor Cocktail III (MedChem Express, Monmouth Junction, NJ, USA). Protein lysates were resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The protein samples were then transferred onto polyvinylidene fluoride (PVDF) membranes. Each membrane was washed in tris-buffered saline with Tween 20 (TBST) thrice, blocked with 5% bovine serum albumin (BSA) blocking-buffer (Beyotime Biotechnology, Shanghai, China) for 1 h at room temperature, and incubated at 4 °C overnight with one of the following rabbit anti-rat primary antibodies: anti-CXCR4 (1:800; Abcam, Cambridge, UK); anti-Arg-1 (1:1,000, Abcam, UK); anti-CD206 (1:500, Boster Biological Technology Company, Wuhan, Hubei, China), or anti-GAPDH (1:1,000; Abcam, UK). This was followed by incubation with horseradish peroxidase (HRP)-conjugated affinity goat anti-rabbit secondary antibody (1:5,000 MultiScience, Hangzhou, Zhejiang, China) for 1 h at room temperature. Immunoreactivity was detected using chemiluminescence reagents (Boster Biological Technology Company, Wuhan, Hubei, China).