Bolt 8 bis tris plus 1.0 mm 12 well precast protein gels

For sumoylation and ubiquitination assays 2 × 10⁶ HEK293T cells were plated in 10 cm dishes and transfected with a total of 7.5 μg of DNA the following day. A modified version of “Detection of Sumoylated Proteins by Immunoprecipitation Analysis”^{61 (link)} was used. Briefly, 16 h post-transfection cells were lysed in SDS lysis buffer (1% SDS, 0.15 M Tris-HCl pH 6.8 and 30% Glycerol). Lysates were boiled for 5 min and diluted 10 × in PBS-T (PBS and 1% Triton-X 100) supplemented with PR-619 (50 μM) and complete protease inhibitors. Cell lysates were sonicated for 20 s then clarified at 4 °C at 13,000 rpm for 10 min. A 20 μl of α-HA beads (SIGMA) or α-FLAG M2 beads (SIGMA) was rotated with cleared protein lysates at 4 °C for 4 h. Furthermore, 4× washes in PBS-T, supplemented with PR-619 (20 μM) and complete protease inhibitors, were performed, and samples eluted by boiling in 60 μl 1× SDS loading dye. Samples were separated by SDS-PAGE using Bolt 8% Bis-Tris plus 1.0 mm 12 well precast protein gels (Life Technologies) in MOPS buffer. Where necessary, the modification smears were quantified using Image Lab Software (Bio-Rad) and normalised to total immunoprecipitated NLRP3 in biological triplicate. A two-tailed Student’s t-test was performed on log2-transformed fold change compared to WT NLRP3 using Prism 7, Graphpad.

NLRP3 sumoylation was assayed by immunoprecipitating NLRP3 from N1-8 BMDMs using α-FLAG M2 beads (SIGMA) as described previously^{15 (link)}. Furthermore, 10 mM N-ethylmaleimide was replaced with 20 μM PR-619, and immunoprecipitates were separated by SDS-PAGE using Bolt 8% Bis-Tris plus 1.0 mm 12 well precast protein gels (Life Technologies) in MOPS buffer.