Hpa01206

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.

Samples were cut into 7 μm thin slices, and cryo-sections were fixed in acetone. After blocking in 4% appropriate serum, sections were incubated with primary antibodies (Mac3 (1:200, M3/84, BD Pharmingen), PARP14 (1:50, HPA01206, Sigma-Aldrich), PARP9 (1:100, ab53796, Abcam) and human CD68 (1:200, M0876, Dako), followed by biotin-labelled secondary antibody (1:250, Vector Laboratories, Burlingame, CA, USA) and streptavidin-coupled Alexa Fluor 488 antibody (Life Technologies). For immunofluorescence double labelling, after avidin/biotin blocking (Vector Laboratories), the second primary antibody was applied overnight at 4 °C, followed by biotin-labelled secondary antibody and streptavidin-coupled Alexa Fluor 594 antibody (1:250, Life Technologies). Sections were washed in PBS and embedded in mounting medium containing 4,6-diamidino-2-phenylindole (Vector Laboratories). For bright-field immunohistochemistry on tissue sections, following the first biotin-labelled secondary antibody incubation, sections were incubated with streptavidin-labelled horseradish peroxidase (HRP) solution (Dako), followed by 3-amino-9-ethylcarbazole (AEC) solution. Slides were examined using the Eclipse 80i microscope (Nikon, Melville, NY, USA) or the confocal microscope A1 (Nikon). All images were processed with the Elements 3.20 software (Nikon).