Protein phosphatase inhibitor

Mouse macrophages were isolated and seeded at 1 × 10⁶ cells/mL in 10 cm dishes and incubated at 37 °C and 5% CO₂ overnight. After pyroptosis induction, the cells were washed with PBS three times and lysed in 500 μL of NP-40 IP lysis buffer containing 1% protein phosphatase inhibitor (Abcam) on ice. Cells were collected and centrifuged at 12,000× g rpm and 4 °C for 15 min. The supernatant was transferred into new tubes and incubated with primary antibodies for 10 h at 4 °C. Dynabeads^™ Protein G for immunoprecipitation (Invitrogen) was added to each tube and incubated at RT for 15 min. After incubation, the magnetic bead-Ab-Ag complex was gently washed 5 times, the supernatant was removed, and the bead-Ab-Ag complex was lysed in SDS sample buffer, boiled for 5 min, and analyzed by SDS-PAGE.

A total of 15 MT were added to RIPA lysis buffer supplemented with a complete protease inhibitor mixture and a protein phosphatase inhibitor (Abcam, UK). For tissue homogenisation, pre-filled bead mill tubes (Thermo Fisher, UK) and an evolution homogeniser was used (Percellys, France). The samples were thoroughly mixed and stored on ice for 10 min before centrifugation at 10,000 g for 10 min. The supernatants were transferred to a fresh tube and centrifuged for further 10 min period. The protein concentrations were measured utilising a Coomassie Plus Bradford assay reagent (Thermo Scientific, UK). The supernatants were stored at − 80 °C.