α pp38

Cells were lysed in RIPA buffer (150 mM NaCl, 10 mM Tris, pH7.4, 0.1% SDS, 1% Triton X-100, 5 mM EDTA, 1% Na-deoxycholate, 1 mM PMSF (phenylmethylsulfonyl fluoride), 1 μg/mL leupeptin, 1 μg/mL aprotinin, 1 μg/mL pepstatin). The primary antibodies used were α-Flag (Sigma-Aldrich, F4042, 1:2000 or Sigma-Aldrich, F7425, 1:1000), α-HA (BioLegend, #901501, 1:1000), α-p38 (Cell signaling, #9212, 1:1000), α-pp38 (Cell signaling, #9219, 1:1000), α-MKK3 (Cell signaling, #5674, 1:500), α-MKK6 (Abnova, #M02, 1:500), α-MAPKAPK2 (Cell signaling, #12155, 1:1000), α-PRMT1 (Sigma-Aldrich, #P16220, 1:1000), α-GAPDH (Cell signaling, G9545, 1:10000), α-mono- and di-methyl arginine (abcam, ab412, 1:500). The secondary antibodies used were anti-mouse (Sigma-Aldrich) or anti-rabbit (Genetex) IgG linked-horseradish peroxidase.

Anti-mouse CD16/32 antibody was used to block the nonspecific binding to Fc receptors before all surface stainings. Dead cells were stained with live and dead violet viability kit (Invitrogen), and were gated out in analysis. Antibodies used for regular flow cytometry are listed in Supplementary Table 1. α-p-p38 was from Cell signaling Technology. For nuclear stainings, cells were fixed and permeabilized using a Mouse Regulatory T Cell Staining Kit (Thermo Fisher Scientific). BD Cytofix/Cytoperm™ kit was used for detection of GM-CSF production. In brief, cells were stimulated by PMA (50 ng/ml, Sigma) and ionomycin (500 ng/ml, Sigma) for 4 h, except for experiments using compound inhibitors in Fig. 5 and S5, where PMA and ionomycin stimulation were not used. Brefeldin A (2 µg/ml, Sigma) was added for the last 2 h before cells were harvested for analysis. For detection of p-p38, cells were first fixed with the BD Cytofix kit and permeablized with 100% methanol followed by staining with rabbit–anti-mouse–p-p38 and APC-goat–anti-rabbit–IgG. Flow-cytometry data were collected using the Gallios flow cytometer (Beckman). Sorting of cells were performed on the Moflo Astrios cell sorter.

The GSK’963 RIPK1 kinase inhibitor was provided by GSK. The following antibodies were used: α-RIPK1 (BD Biosciences, 610459), α-HOIL (gift from Henning Walczak), α-cIAP1 (Enzo, ALX-803-335-C100), α-TNFR1 (Abcam, 19139), α-Actin (Santa Cruz Biotechnology, sc-1615), α-P-p65 (Cell Signaling, 3033), α-p65 (Cell Signaling, 8242), α-IkBα (Santa Cruz, sc-371), α-P-p38 (Cell Signaling, 9215), α-p38 (Cell Signaling, 9212), α-P-JNK (Cell Signaling, 9255), α-JNK (Santa Cruz Biotechnology, sc-571), α-P-ERK (Cell Signaling, 9101), α-ERK (gift from Chris Marshall) α-caspase-8 (Cell Signaling, 9429), α-FLAG [M2] (SIGMA, M8823), α-Ub (Dako, Z0458), α-FLIP (Adipogene, AG-20B-0056), α-FADD (Santa Cruz Biotechnology, sc-6036), α-RIPK3 (ProSci, 2283), α-Tubulin (SIGMA, T9026), α-SHARPIN (Proteintech, 14626-1-AP), α-TRAF2 (Cell Signaling, 4712), α-CD8-PE-Cy7, GR-1-PE-Cy7, CD11c-FITC, CD4-FITC, CD11b-Cy5, B220-FITC (gift from Henning Walczak), α-CD69-PE (eBioscience, 12-0691-82), α-CD3-APC (eBioscience, 47-0032-82), and α-CD16 (eBioscience, 14-0161-82).