Ab288437

The protein expression pattern of Galectin-9, CD163 and CXCR3 in both THP-1_NC and LGALS9_OE cells was evaluated by western blotting. Briefly, cells were seeded in six-well plates and cultured to 70% confluence. Then, whole cell lysates were extracted and prepared with RIPA buffer containing protease inhibitor cocktail. Western blotting was conducted with 100 µg of the protein extract as described elsewhere [19 ]. Western blot was performed with anti-β-actin (1:1000, TA09, Zhongshan Goldenbridge Biotechnology, Beijing, China), anti-CD163 (1:500, TA506381, Zhongshan Goldenbridge Biotechnology, Beijing, China), anti-Galectin9 (1:200, ab184331, Abcam, Cambridge, UK) and anti-CXCR3 (1:500, ab288437, Abcam, Cambridge, UK).

Total protein in the cells was extracted using RIPA lysis buffer (89901, Invitrogen) and quantified using a BCA kit (23225, Invitrogen). Total protein (20 µg) was separated using 10% SDS-PAGE and transferred to a polyvinylidene fluoride membrane (Millipore, Bedford, USA). The membrane was blocked with 5% nonfat milk and incubated with primary antibodies overnight at 4°C. The membranes were then incubated with the corresponding secondary antibodies for 2 h at room temperature. The protein bands were detected using the ECL detection kit (Millipore). The signal intensity was analyzed using ImageJ software. The following primary antibodies were used: anti-CCR4 (1:1,000, ab254376, Abcam, Cambridge, UK), anti-CXCR3 (1:1,000, ab288437, Abcam, Cambridge, UK), anti-P2RY14 (1:1,000, ab136264, Abcam, Cambridge, UK), anti-CCR2 (1:1,500, #12199, Cell Signaling Technology, Massachusetts, USA), anti-CCR8 (1:1,000, ab32399, Abcam, Cambridge, UK), and anti-CCL19 (1:1,000, ab192877, Abcam, Cambridge, UK).

Tumours from each group were minced and lysed in lysis buffer for immunoblotting (P0013, Beyotime biotechnology) with deacetylase inhibitor cocktail (P1113, Beyotime biotechnology). Proteins were separated in SDS polyacrylamide gel, electro-transferred onto nitrocellulose membranes (FFN08, Beyotime biotechnology), and blocked-in blocking buffer (P0023B, Beyotime biotechnology). Then, the membranes were incubated with different antibodies overnight: Ifn-γ (15365-1-AP, Proteintech, 1:2000 dilution), Cxcl9 (701117, Invitrogen, 1:500 dilution), Cxcr3 (ab288437, Abcam, 1:1000 dilution), Acetyl-histone H3 (8173 T, CST, 1:1000 dilution), Histone H3 (4499 T, CST, 1:3000 dilution), Pdl-1 (66248-1-Ag, Proteintech, 1:5000 dilution), β-actin (4970 T, CST, 1:1000 dilution). Corresponding secondary antibodies at room temperature for 2 h. The protein bands were visualized in Imagequant 800 system.