Ripk1 antibody

MPEC cells were cultured at 1 x 10⁶ in 15 cm plates with and without shRNA for 72 hours at 37°C with 5% CO₂. A higher concentration of TRAIL was used for shorter time points to increase the speed of necrosome formation. Cells were treated with 500 ng/mL TRAIL for 2 hours and lysates harvested in IP buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate, and 1 mM β-glycerolphosphate) with phosphatase inhibitors. Lysates were spiked with 8 μg of RIPK1 antibody (BD Transduction Lab, 610459) and incubated overnight at 4°C. Protein A/G agarose beads (Santa Cruz, #sc-2003) were added and incubated for 2 hours and washed with IP buffer at least three times. Samples were run on a polyacrylamide gel and immunoblotted.

Primary antibodies for cell staining included the following: RIPK1 antibody (610458; BD Biosciences) and RIPK3 antibody (sc135170; Santa Cruz Biotechnology). Secondary antibodies, including Alexa Fluor 488-conjugated anti-mouse IgG and Cy3-conjugated anti-rabbit IgG, were provided by the Center for Biologic Imaging, University of Pittsburgh. PE-Annexin V 7-AAD Apoptosis Detection Kit (559763) was purchased from BD Biosciences. CM-H2DCFDA (C6827) was from Thermo Fisher Scientific. Anti-mouse Ly-6G FITC (11–5931-82), anti-mouse Ly-6G PE-Cyanine5 (15–5931-81), anti-mouse CD11b PE (12– 0112-81), and anti-mouse F4/80 antigen FITC (11–4801-81) were purchased from Thermo Fisher Scientific. PE anti-mouse CD63 antibody (143903) was from BioLegend (San Diego, CA, USA). Anti-mouse Ly-6G and Ly-6C particles (558111) were purchased from BD Biosciences. Necroptosis inhibitor Nec-1 (11658) was purchased from Cayman Chemical (Ann Arbor, MI, USA). Exosome-releasing inhibitor DMA (sc-202459) was purchased from Santa Cruz Biotechnology. N-Acetyl-l-cysteine (A7250) was purchased from Sigma-Aldrich. Dil cell-labeling solutions (V22885), total exosome isolation reagent (4478359), and exosome-depleted FBS (A2720801) were purchased from Thermo Fisher Scientific.