Phospho rip3

Dexamethasone (#D4902) and 2,4-dinitrochlorobenzene (DNCB, #138630) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Castor oil and acetone were purchased from Aladdin Chemical (Shanghai, China) and Sinopharm Chemical Reagent (Shanghai, China), respectively. Propidium iodide (PI) / RNase staining solution (#4087 S) was purchased from Cell Signaling Technology (Danvers, MA, USA). In this study, some antibodies were purchased from Cell Signaling Technology, including phospho-RIP (#65746 for human and #38662 for mouse), RIP3 (#95702), phospho-RIP3(#91702), MLKL (#37705), phospho-MLKL (#37333), GAPDH (#5174), and goat anti-rabbit IgG HRP-linked antibody (#7074). Other antibodies were purchased from Abcam (Cambridge, MA, USA), including RIP (#ab125072, #ab72139), RIP3 (#ab226297), phospho-RIP3 (#ab209384), MLKL (#ab184718), phospho-MLKL (#ab187091), CD4 (#ab183685), CD8 (#ab217344), MPO (#ab208670), MMP-9 (#ab283575) and NE (#ab68672). Tumor necrosis factor α (TNF-α), Smac-mimetic compound, z-VAD-fmk and necrostatin-1 (as described previously [27 (link)]) were gifts from Dr. Sudan He’s Lab in Suzhou Institute of Systems Medicine. TNF-α and IFN-γ used to simulate the ACD microenvironment in HaCaT were purchased from R&D system (Minneapolis, USA) and PeproTech (New Jersey, USA). rHMGB1 was purchased from R&D system (Minneapolis, USA).

The histone deacetylase (HDAC) inhibitor SAHA was purchased from Selleckchem (Houston, TX, USA), the MDM2 inhibitor RG7388 was purchased from MedChemExpress (Monmouth Junction, NJ, USA). Letrozole was purchased from Selleckchem (Houston, TX, USA), and Necrostatin was purchased from Abcam (Cambridge, MA, USA). The primary antibodies against p53, phsoph-p53, p21, CDC25C, TIPM-1, CDK1, BAK, BAX, APAF1, Bcl-XL, RIP1, and cleaved PARP (1:1000) were purchased from Cell Signaling Technology (Danvers, MA, USA). Phospho-RIP3 and MLKL (1:1000) antibodies were purchase from Abcam (Cambridge). MDM2 (1:500) was purchased from Santa Cruz biotechnology (Dallas, TX, USA). The β-actin antibody (1:2000) was purchased from Sigma Aldrich chemical company (St. Louis, MO, USA). The secondary antibodies anti-rabbit, anti-mouse, HRP conjugated, and DMSO were purchased from Sigma Aldrich chemical company. Nitrocellulose membranes (0.45 µm) were purchased from Amersham (GE Healthcare Life Sciences, Marlborough, MA, USA). ECL was purchased from KPL biosolutions (Milford, MA, USA). SYTOX^® Green and DEVD-amc CellEvent^TM Caspase-3/7 Green ReadyProbe^TM were purchased from Thermo Fisher (Molecular Probes, Life Technologies, Carlsbad, CA, USA). All other chemicals used in this study were of research grade.