Cst lysis buffer

Cells were lysed with RIPA buffer (Sigma R0278) supplemented with additional 2% NP-40. Cell lysates were sonicated before protein concentration determination (BCA protein assay kit, Pierce 23225). Equal amount of protein was fractionated by SDS-polyacrylamide gel eletrophoresis and transferred onto a polyvinylidene difluoride membrane, which was blocked with 5% milk in Tris-buffered saline (TBS) followed with desired antibodies in 5% BSA-TBS. The protocol for co-immunoprecipitation has been described previously^{58 (link)}. Briefly, cells were lysed with Cell Signaling Technology (CST) lysis buffer (CST9803) supplemented with 1 mM phenylmethylsulfonyl fluoride and incubated with specific antibodies with protein A/G-Sepharose overnight. After three times washing with CST lysis, the precipitated proteins were eluted with SDS-loading buffer and analyzed by immunoblotting. For the ubiquitination assay, cells transfected with plasmids were lysed with RIPA buffer supplemented with additional 0.1% SDS to a final concentration of 0.2% SDS, followed by standard immunoprecipitation protocols. Chemiluminescence was detected with X-Ray films or Fusion FX (Analis). Image J software was used to quantify the immunoblots by densitometry. Information on the antibodies used is presented in Supplementary Dataset 2.

Cells were lysed in CST lysis buffer (Cell Signaling Technology) supplemented with MG132 (40 μM; Calbiochem) and cOmplete Protease Inhibitor Cocktail (Roche). Lysates were separated by SDS–PAGE and immunoblotting performed on polyvinylidene difluoride transfer membranes (Fisher Scientific) with primary antibodies diluted in 5% BSA overnight at 4 °C. The following antibodies were used for immunoblotting: GLUT1 (1:1,000; Alpha Diagnostic; GT11-A), p63 (1:1,000; Biocare Medical; CM163A), CK5 (1:1,000; Abcam; ab52635), HIF-1α (1:1,000; BD Transduction Laboratories; 610959), AKT and Ser473-p-AKT (1:1,000; Cell Signaling; 9,272 and 9,271, respectively), β-actin (1:5,000; Sigma; A5441). Horseradish peroxidase-conjugated secondary antibodies (Santa Cruz) were diluted 1:5,000 in 5% skim milk, followed by detection with SuperSignal West Femto or Pico substrate kits (ThermoFisher). Uncropped images of immunoblots are provided as Supplementary Fig. 22.

LX-2 cells were harvested after 24 h incubation time at either 1 or 10 % FBS at ~85 % confluency using trypsin-EDTA. Cells were lysed in CST lysis buffer (Cell Signaling Technologies, Danvers, MA, USA) supplemented with protease inhibitor cocktail (Sigma Aldrich). SDS-PAGE (NuPage, Thermo Fisher, Waltham, MA, USA) was used to resolve equal amounts of protein. Semi-dry blotting was performed onto nitrocellulose membranes (Amersham, GE Healthcare, Chicago, IL, USA). Blocking of membranes was achieved by 5% skim milk in TBS-T for 30 min at RT. Primary antibody (α-SMA, Invitrogen 1A4; vinculin, Invitrogen 7F9) incubation was performed over night at 4 °C and secondary HRP antibody (horse anti-mouse 7076, Cell Signaling Technologies) incubation was performed at RT for 1 h. Chemiluminescent detection agent Pierce ECL PLUS (Thermo Fisher) was used as substrate and detection was done at a ChemiDoc MP (BioRad, Hercules, CA, USA). Image analysis was carried out with Image Lab (BioRad).

BMDCs, peritoneal macrophages, or peritoneal neutrophils were lysed with 1X CST lysis buffer (Cell Signaling). For neutrophils, DFP (Sigma Aldrich) was added to the lysis buffer to inhibit any protease activity. BCA assay kit (Thermo Fisher) was used to determine protein concentration from lysates. Twenty μg of protein from lysates mixed with 1X SDS (from 5X stock), and Ultrapure water (Invitrogen) were boiled for 10 minutes at 95°C on a heating block. Samples were loaded into 4%–20% mini-PROTEAN, 10-well, 50 μl TGX precast SDS-PAGE gels (Bio-rad). Gels were run in 1X TAE buffer at a constant 110V. Proteins were transferred onto a nitrocellulose membrane using Bio-rad Trans-blot Turbo transfer system. The membrane was blocked with 5% milk for 1 hour at RT, and incubated with rabbit anti-mouse GSDMD (EPR20859, Abcam) or mouse anti-β-actin (Santa Cruz Biotechnology) diluted in 5% milk and incubated at 4°C overnight on a rocker. Membranes were washed with 1X TBST buffer 3× for 10 minutes. HRP-conjugated secondary antibodies against rabbit or mouse IgG (Cell Signaling) were diluted in 5% milk and incubated at RT for 1 hour. West Femto Maximum Supersignal (Thermo Fisher) was used to enhance signal before the membrane was imaged by the Chemidoc (BioRad) instrument.