Anti caspase 5

The anti-FLAG and anti-caspase-11 antibodies were from Sigma. The anti-SREBP1, anti-HMGCS1, anti-HMGCR antibodies were from Santa Cruz Biotechnology. The anti-HA antibody was from Roche, and the anti-S1P and anti-GAPDH antibodies were from Abcam. The anti-RCAS1, anti-EEA1, anti-LAMP1, anti-human caspase-4 and anti-caspase-5 antibodies were from Cell Signaling Technology. The LPS was purchased from Sigma (L2637).

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.

Immunofluorescence staining was performed on 8 μm serial frozen sections of healthy and psoriatic skin fixed for 5 minutes in acetone at -20°C. The sections were blocked in 10% normal goat serum, and incubated overnight with anti-caspase-5 (Cell Signaling Technology, Inc., Beverly, MA, USA; 1:50). The sections were then incubated with goat anti-rabbit IgG conjugated with Alexa Fluor^® 647 (Invitrogen, Carlsbad, CA, USA; 1:250), diluted in 10% normal goat serum and incubated for 1 hour at room temperature in a dark humidified chamber. Staining with secondary antibodies only was performed as a negative control. Sections were overlaid with ProLong Gold antifade reagent containing DAPI (Invitrogen, Carlsbad, CA, USA). Fluorescent stained tissues were imaged using a fluorescent microscope Zeiss ImagerZ1 (Zeiss, Jena, Germany) using a 12-bit CCD digital camera PCO PixelFly (PCO, Kelheim, Germany).