Sds page gel electrophoresis

Cells were harvested and lysed in radioimmunoprecipitation (RIPA) buffer as previously described [54 (link)]. Equal amounts of proteins were separated by SDS-PAGE gel electrophoresis (Bio-Rad). Proteins were transferred to an Immobilion-P transfer membrane (Merck Millipore), and detected using standard immunoblotting techniques. Proteins were visualized using SuperSignal™ West Dura Extended Duration Substrate kit (Thermo Fisher Scientific) according to the manufacturer's instructions. Images were captured using a Syngene ChemiGenious camera and presented by the GeneSnap software tool (Syngene, Cambridge, England). Intensity of protein bands was quantified by using the GeneTool software (Syngene).

Tissues and cells were lysed in RIPA buffer (Beyotime, China). 40 μg of protein was used for SDS-PAGE gel electrophoresis (Bio-Rad) and transferred onto PVDF membranes (Millipore, China). Blocking was performed with 5% milk, and then the membranes were incubated with primary antibodies. Anti-NAA25 (1:1, 000 HPA039322, Sigma-Aldrich) or anti-actin (1:5000, A1978, Sigma-Aldrich) was added and incubated overnight at 4°C. After being washed, the membranes were incubated with secondary antibodies (peroxidase conjugated, suitable for each primary antibody) for 2 hours at room temperature. The signal was detected with a Bio-Rad ChemiDoc XRS + System after adding Super Signal West Pico chemiluminescence.

Samples were separated by SDS-PAGE gel electrophoresis (BioRad) for in-gel reduction with 10 mM DTT, alkylation with 20 mM IAA and trypsin digestion (1:50 enzyme:protein) overnight at 37 °C (V5111, Promega). Extracted peptides were lyophilized and then resuspended in 0.1% formic acid (FA) for desalting using C18 OMIX tips (A57003100K, Agilent). Samples were lyophilized again and resuspended in 0.1% FA, bath sonicated for 20 min, and then centrifuged at 14,000xg for 15 min to remove insoluble debris, and the supernatants (clarified peptides) were aliquoted into vials and then analyzed by LC–MS/MS.

Detection of ASC oligomers, NLRP3 and cleaved caspase-1 and IL-1β was detected using western blot analysis as described in Låg et al. [80 (link)]. Briefly, samples for western blot were suspended in laemmli sample buffer consisting of 10% glycerol (Merck, Darmstadt, Germany) 5% mercaptoethanol (Sigma, St. Louis, MO, USA), 2% sodium dodecyl sulphate (SDS) (Sigma, St. Louis, MO, USA) and 0.01% bromophenol blue (Sigma, St. Louis, MO, USA), and incubated at 95 °C for 10 min to denature the proteins. The proteins were then separated using 8 and 15% SDS-PAGE gel electrophoresis (BioRad, Hercules, California, USA) and transferred to a nitrocellulose membrane (Maine Manufacturing LLC, Sanford, ME, USA). The membranes were incubated overnight with specific antibodies against ASC (Santa Cruz Biotechnology, Dallas, Texas, USA.), IL-1β (Cell Signalling Technology, Danvers, Massachusetts, USA) and caspase-1 (Cell Signalling Technology, Danvers, Massachusetts, USA) 3% milk or in 5% bovine serum albumin (BSA). Next, the membranes were incubated with HRP-conjugated anti-mouse or anti-rabbit secondary antibodies (Dako, Santa Clara, Ca, USA) and SuperSignal West Dura (Thermo scientific, Waltham, MA, USA). Chemiluminescence was monitored and recorded using a Chemi-Doc with Image lab software (Both from Bio-Rad, Hercules, CA, USA).