Ab54461

Peritoneal macrophages were washed with ice‐cold PBS and homogenized on ice using RIPA lysis buffer (Beyotime) supplemented with complete protease inhibitor (Beyotime). Samples were loaded on 10% SDS‐PAGE and transferred onto PVDF membranes (Bio‐Rad, Shanghai, China). Membranes were blocked with 5% non‐fat dry milk and incubated with primary antibodies against iNOS (ab129372; Abcam, Cambridge, USA), Arg‐I (ab91279; Abcam), p‐STAT6 (ab54461; Abcam), STAT6 (ab32520; Abcam), respectively. β‐actin was used as control. The intensities of the corresponding protein bands were evaluated by densitometry via Image J (National Institutes of Health, USA) Protein expression was assessed relative to β‐actin or indicated protein.

Cells were lysed with RIPA buffer containing protease inhibitor (Roche). Protein concentration was measured using the bicinchoninic acid method (Thermo Scientific). Total protein was separated by 8–10% SDS–PAGE and transferred using the iBlot western blotting system (Life Technologies). Primary antibodies against human and mouse PARP14 (1:250, HPA01206 Sigma-Aldrich), human and mouse PARP9 (1:250, ab53796, Abcam), human and mouse STAT1 (1:1,000, #9172, Cell Signaling), phosphorylated STAT1 (1:1,000, #9167, Cell Signaling), human and mouse STAT6 (1:2,000, #9362, Cell Signaling), mouse (1:1,000, ab54461, Abcam) and human (1:2,000, #9361, Cell Signaling) phosphorylated STAT6 and human and mouse β-actin (1:5,000; Novus) were used. For secondary antibodies, we used anti-mouse (1:1,000–5,000, A4416, Sigma) and rabbit (1:1,000–5,000, NA934-1ML, GE Healthcare Life Sciences) IgG antibodies. Protein expression was detected using Pierce ECL Western Blotting substrate reagent (Thermo Scientific) and ImageQuant LAS 4000 (GE Healthcare). Uncropped images of western blots are demonstrated in Supplementary Figs 18–20.

The following antibodies were purchased from Cell Signalling Technology: pSTAT1 (#8826), Arginase‐1 (#9819), Rab7 (#9367), Rab5 (#2143), BIP (#3183), EEA1 (#2411), IκBα (#9242), LAMP1 (#3243), Na⁺/K⁺ ATPase 1 (#3010), Lamin A/C (#2032), TAK1 (#4505), JNK (#9258), p‐JNK (#9251), p‐c‐Jun (#9165) and Vimentin (#5741). Antibodies purchased from Abcam were as follows: pSTAT6 (ab54461), CD36 (ab133625), CD14 (ab182032) and MSR1 (ab151707, ab79940). Antibodies against K63‐specific ubiquitin (#05‐1308) and ITGAM (PAB12135) were from Millipore and Abnova, respectively. Sheep antibodies against MSR1, TAK1, TAB1, TAB2, MKK7 and MKK4, and rabbit IgG were generated by the Antibody Production Team of the Division of Signal Transduction Therapy (DSTT), Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, United Kingdom. The antibody used for MSR1 immunohistochemistry was clone SRA‐E5 (from Abnova, #MAB1710). Commercial antibodies were used according to the manufacturer instructions. DSTT‐made antibodies were used at 2 μg/ml in TBS‐T containing 5% non‐fat‐dried milk. Recombinant proteins, plasmids and antibodies generated for the present study are available to request on our reagents website (https://mrcppureagents.dundee.ac.uk/).