P tbk1

Mouse monoclonal antibodies against Flag (Sigma), HA (Origene), β-actin (Sigma), p-IκBα (CST), p-IRF3 (CST) and p-TBK1 (Abcam); poly(I:C) (Invivogen), LPS (Sigma), R848 (Invivogen), PGN (Invivogen), human IFN-γ (PeproTech), Bafilomycin (Sigma), Monophosphoryl lipid A (Sigma), Z-KAD-FMK (MCE) were purchased from the indicated companies. Luminescent cell viability assay kit (G7570) was purchased from Promega. Mouse antisera to TRIM32 and TRIF were raised against recombinant human TRIM32 and murine TRIF(1–475) respectively. Rabbit antisera to TAX1BP1 and NDP52 were raised against recombinant murine TAX1BP1 and NDP52(1–160) respectively.

Tissue lysates were homogenized in SDS lysis buffer [Cell Signaling Technology (CST), #7722] and transferred onto polyvinylidene difluoride membranes. After blocking in 5% milk in tris-buffered saline and 0.1% Tween 20, membranes were incubated overnight with the following antibodies at 4°C: CASP9 (Abcam, #ab202068), cleaved CASP3 (CST, #9664), RIP3 (Millipore Sigma, #PRS2283), BAX (CST, #2772), BCL2 (CST, #3498), GSDMD (Santa Cruz Biotechnology, sc-393656), NLRP3 (CST, #15101), ACSL4 (Abcam, #ab155282), LC-3 (CST, #2775S), cGAS (CST, #31659), STING (CST, #13647S), TBK1 (CST, #38066), pTBK1 (CST, #5483), IRF3 (CST, #4302), pIRF3 (CST, #4947), p65 (CST, #8242), pp65 (CST, #3033), and GAPDH (CST, #5174). Membranes were incubated with horseradish peroxidase–conjugated secondary anti-rabbit or anti-mouse antibody (CST, #7074 and #7076) for 1 hour at room temperature. Signal was detected by enhanced chemiluminescence (SuperSignal West Femto Maximum Sensitivity Substrate, #34094; Thermo Fisher Scientific). Western blots were quantified using the Fiji software (58 (link)).

Poly(I:C) HMW (Invivogen Cat# tlrl-pic); IL-1β (PEPROTECH 200-01B Cat# tlrl-pic); Peptidoglycans (PGN) (SIGMA Cat#77140 Peptidoglycan from Staphylococcus aureus); TNF-α (PEPROTECH Cat# 300-01 A); Doxorubicin (hydrochloride) (MCE Cat#HY-15142); Staurosporine (MCE Cat#HY-15141); Thapsigargin (SIGMA Cat# T9033) Etoposide (Selleck Cat#33419-42-0); Caspase inhibitor z-VAD-FMK (Beyotime Cat#C1202); Caspase 3 inhibitor Ac-DEVD-CHO (Beyotime Cat#C1206); ELISA kit for murine IFN-β, TNFα, IL-6 (BioLegend Cat#439408, Cat#430907, Cat#431307); PARP(46D11), phospho-NF-κB p65(Ser536), Phsopho-IRF-3 (Ser396) monoclonal Antibody (CST Cat#9532, Cat#3033, Cat#4947); IRF3, TBK1, RELB, c-Rel Polyclonal Antibody (ABclonal Cat#A11118, Cat#A2573, Cat#A0519, Cat#A1181); p65 RELA polyclonal Antibody (ProteinTech Cat#10745-1-AP); p-TBK1(phosphor S172), IKKi/IKKε antibody (Abcam Cat#ab109272, Cat#ab124766); Caspase-3(E-8) (Santa Cruz Cat#sc-7272). GSTSep Glutathione Agarose Resin (Yeasen 20507ES10). Caspase-Glo 3/7 Assay (Promega G8090). Protein A/G Magnetic Beads (Bimake B23202)