Ab109457

Total cellular proteins were isolated using RIPA buffer (Beyotime, Shanghai, China) and quantified with a BCA Protein Assay Kit (Beyotime). Proteins (40 µg per sample) were loaded in each lane and separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) before being transferred to polyvinylidene fluoride (PVDF) membranes (Millipore) via the semi-dry transfer system (Bio-Rad). The membranes were washed with double distilled H₂O (ddH₂O) and blocked with 5% skim milk in PBS with 0.05% Tween-20 (PBST) for 30 min at room temperature, and then exposed to a primary antibody overnight at 4 °C. The primary antibodies were as follows: CALD1 (1:10,000, ab32330, Abcam, USA), PD-L1 (1:1,000, #13684, CST); p-JAK1 (1:1,000, ab13805, Abcam); JAK1 (1:1,000, ab133666, Abcam); p-JAK2 (1:1,000, #3771, CST); JAK2 (1:1,000, 3230, CST); p-STAT1 (1:1,000, ab109457, Abcam); and STAT1 (1:1,000, ab234400, Abcam). Endogenous GAPDH (1:20,000, #5174, CST) was performed as an internal standard for WB analysis. On the following day, after washing with tris-buffered saline with 0.1% Tween-20 (TBST) thrice for 10 min each time, the membranes were incubated with secondary antibodies for 1 h at room temperature and then visualized and quantified using the Odyssey two-color infrared laser imaging system (LI-COR Biosciences, Lincoln, NE, USA).

Cells were washed twice with cold PBS, collected using cell scrapers and transferred to pre-chilled tubes. Collected cells were centrifuged at 500g for 5 min, and cell pellets were stored at −80 °C. Proteins were extracted from cell pellets using a lysis buffer for western blotting and their concentrations were measured by bicinchoninic acid assay (Applygen Technologies, P1511). Western blotting was performed according to standard procedures [25 (link)]. The primary antibodies were anti-PLXND1 (NBP1-33634, Novus), anti-SEMA3E (AF3239, R&D Systems), anti-iNOS (ab283655, Abcam), anti-CD206 (ab125028, Abcam), anti-CD163 (ab156769, Abcam), anti-Liver Arginase (ab133543, Abcam), anti-STAT1 (phospho Y701) (ab109457, Abcam), anti-STAT1 (ab109320, Abcam), anti-STAT6 (phospho Y641) (ab263947, Abcam), anti-STAT6 (ab32520, Abcam) and anti-GAPDH (ab9485, Abcam) acted as an internal control. Western blots were quantified using the FIJI software. Uncropped Western blot gels are available in the Supplementary Information section.