Anti total syk

Total cell lysates were prepared using Laemmli buffer containing 5% β-mercaptoethanol. Protein concentrations were determined using Nanodrop ND1000 (Thermo Scientific). Proteins (20–50 μg) were boiled, separated on SDS–PAGE, and transferred onto PVDF membranes (Bio-Rad Laboratories). Membranes were blocked with 5% non-fat dry milk in phosphate-buffered saline (PBS) with 0.1% Tween 20 (PBST, Sigma-Aldrich) for 1 h at room temperature, and incubated overnight at 4 °C with the following primary antibodies diluted 1:1,000 in 5% BSA/PBST; anti-NLRP3 (#AG-20B-0014-C100, Adipogen), anti-IL-1β (#2022), anti-total Syk (#2712), anti-caspase-1 (#4199), anti-caspase-5 (#4429), anti-caspase-4 (#4450) (all from Cell Signaling) and anti-GAPDH (#MAB374, Millipore). For phospho-Syk detection, membranes were blocked and incubated with the primary antibody (#2710S, Cell Signaling) in 5% BSA in Tris-buffered saline with 0.1% Tween 20 (TBST). Densitometry analysis was performed using ImageJ software and the data were normalized against GAPDH. Images have been cropped for presentation. Full-size blot images shown in Figs 1, 2, 3, 4, 5, 6, 7 and Supplementary Figs 1, 5, 8 and 9 are included in Supplementary Fig. 11.

Immunoprecipitation (IP) products or protein extracts were loaded on a bisacrylamide
gel. Primary antibodies and dilutions were as follows: rabbit anti-phospho Erk (Cell
signaling, 1:1,000), rabbit anti-Total Erk (Cell signaling, 1:1,000), anti-phospho
Syk (Cell signaling, 1:500), anti-Total Syk (Cell signaling, 1:1,000), mouse
anti-phospho IkB (Cell signaling, 1:1,000), rabbit anti-Total IkB (Cell signaling,
1:1,000), mouse anti-Flag-M2 (Sigma, 1/1,000 or 1:20,000), anti-COP1 (Bethyl,
1:1,000), anti-CD72 (Santa Cruz Biotechnology, 1:1,000). The secondary antibodies and
dilution were as follows: donkey anti-rabbit IgG (GE Healthcare, 1:10,000), mouse
anti-rabbit light chain (Abcam, 1:20,000). The ratio phospho-p42/Total-p42 for one
sample corresponds with the ratio between the values of phospho-Erk and total-Erk
band density for that sample. The density of each band was measured with ImageJ
software.