Zbp1 antibody ag 20b 0010 c100

After indicated treatments, cells were lysed in 1X Laemmli Buffer containing 5% β-mercaptoethanol, boiled for 15 min and incubated on ice for 15 min. For indicated conditions, proteins were precipitated from cell supernatants by methanol–chloroform extraction, and precipitated proteins were processed as above prior to loading and running on SDS-PAGE gels. Primary antibodies against Casp8 (8592), Casp3 (9665), Casp7 (9492), Casp9 (9508), Casp1 (24232), Casp11 (14340), RIPK1 (3493), pRIPK1 (Ser166) (31122), pRIPK1 (Ser321) (38662), IL-1β (12242), FLAG (8146), and GAPDH (2118) were purchased from Cell Signaling Technologies. ZBP1 antibody (AG-20B-0010-C100) was purchased from Adipogen. GSDMD antibody (ab209845) was purchased from Abcam. FADD antibody (05-486) was purchased from Millipore Sigma. All primary antibodies were used at a dilution of 1:1000. Secondary antibodies, anti-rabbit IgG (H + L) (DyLight^TM 800 4X PEG Conjugate) (5151), anti-mouse IgG (H + L) (DyLight^TM 800 4X PEG Conjugate) (5257), and HRP-linked anti-rat IgG antibody (7077) were purchased from Cell Signaling Technologies. Fluorescent secondary antibodies were used at a dilution of 1:30,000 and HRP-based secondary antibodies were used at a dilution of 1:2000. Uncropped western blots are available in the Source data file.