Phospho mlkl

Primary antibodies used for immunofluorescence microscopy and Western Blotting were obtained from Cell Signaling Technology and included: Beta-actin (#3700 and #4970), phospho-p70s6k T389 (#9234), NF kappa B p65 (#8242S), beta-tubulin (#2128S), Caspase-3 (#14220), Caspase-1 (#3866), Phospho-MLKL (#91689). Secondary antibodies used to detect the primary antibodies for Western Blotting were obtained from Thermo Fisher Scientific, and included goat anti-rabbit and goat anti-mouse IgG-HRP (#31460 and #31430). Secondary antibodies used for immunofluorescence microscopy were obtained from Molecular Probes (Life Technologies), and included goat anti-rabbit IgG AlexaFluor488 and goat anti-mouse IgG AlexaFluor594.

Cells were lysed using RIPA lysis buffer (Thermo-Fisher Scientific) supplemented with Halt protease and phosphatase inhibitor cocktail (Thermo-Fisher Scientific). Protein concentrations were measured by using a Detergent Compatible protein assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA), and the same amount of protein was loaded into a 4–15% NuPAGE Bis–Tris Protein gel. Gels were transferred using an iBlot Dry blotting system onto nitrocellulose membranes (Thermo-Fisher Scientific), and the transferred samples were blocked for 1 h at RT in 5% non-fat-dry-milk in tris-buffered saline (TBS). Primary antibodies used included: Phospho-MLKL (Cat# 916,895, Cell Signaling, Danvers, MA), Mixed Lineage Kinase Domain Like Pseudokinase (MLKL) (Cat #14993S, Cell Signaling), and B cell lymphoma (Bcl)-2 (Cat#Ab182858, Abcam, Cambridge, UK). Images of chemiluminescent bands were acquired using a Bio-Rad Gel documentation system (Bio-Rad Laboratories, Inc.). Membranes were washed in TBS-T, blotted with anti-tubulin antibody (Abcam CAT#Ab6046), and developed similarly. Image Lab Software version 6.0.1 (Bio-Rad Laboratories, Inc.) was used to perform densitometric analysis. Results were expressed as fold-change compared to control cells.

Protein lysates were extracted using ice‐cold lysis buffer (1% NP‐40, 1mM DTT, supplemented with protease and phosphatase inhibitors in PBS) and subjected to immunoblotting. Primary antibodies against APP and β‐actin were obtained from Biolegend, and Santa Cruz Biotechnology Inc, respectively. RIP, phospho‐RIP, MLKL, phospho‐MLKL, RIP3 and phospho‐RIP3 were purchased from Cell Signalling Technology Inc. The dilution ratio used for each antibody was shown in Table S5. All images were captured using the ChemiDocTM XRS+molecular imager (Bio‐Rad Laboratories).

Cells for Western blot analysis were lysed in Lämmli Buffer (Bio-Rad, Hercules, CA, USA) with protease inhibitors (Thermo Fisher, Waltham, MA, USA) and sodium orthovanadate (Sigma Aldrich, St. Louis, MO, USA) according to the manufacturer’s protocol. Thirty micrograms of total protein were separated on ExcelGels (GE Healthcare) and transferred onto nitrocellulose membranes (Bio-Rad). After blocking, membranes were incubated with primary antibodies [cleaved caspase-3 antibody (0.5 µg/ml, #MAB835; R&D Systems, Minneapolis, MN, USA), phospho-RIPKs 1 (1:100, #65746; Cell Signalling Technology, Cambridge, UK), phospho-RIPK3 (1:200, #ab209384; Abcam, Cambridge, UK), phospho-MLKL (1:500, #91689; Cell Signalling Technology, Cambridge, UK), or glyceraldehyde 3-phosphate dehydrogenase (1:2000, #2118; Cell Signalling Technology, TNF (1 µg/ml, R&D Systems)] overnight at 4 °C. After further incubation with horseradish-conjugated goat-anti-rabbit antibody (1:10,000, #170-6515; Bio-Rad, Hercules, CA, USA), secondary antibodies were visualized with Supersignal West Dura (Thermo Fisher, Waltham, MA, USA) and signals were detected using ChemiDoc System (Bio-Rad). For blocking of the TNF antibody, 1 µg TNF antibody was pre-incubated with 10 µg recombinant TNF (R&D Systems) for 4 h at 4 °C:

Primary antibodies against p53, Phospho-Akt (S473), Total-Akt, PUMA, Phospho-FoxO3a (S253), Total-FoxO3a, Phospho-p65 (S536), Total-p65, Bax, Noxa, Bid, Bim, Bcl-2, Bcl-XL, Mcl-1, Cox IV, Cleaved-Caspase3, β-actin LC3, Total-MLKL, and Phospho-MLKL (Ser358) were purchased from Cell Signaling Technology. Lipofectamine™ Reagent was purchased from Invitrogen. HRP-conjugated anti-rabbit and/or anti-mouse secondary antibodies and ECL-plus kit were from GE Healthcare. Ipatasertib and 5-FU were purchased from APP Pharmaceuticals. afuresertib and perifosine were purchased from Selleck Chemicals(Houston, TX). Regofenib and cisplatin were purchased from Axon Medchem. Other chemicals were mainly from Sigma. CCK-8 kit was from 7 Sea Biotech (Shanghai, China). The plasmid of expressing PUMA was kindly supplied by Jian Yu, PhD^{9 (link)}.

Human umbilical cord MSCs (hUC-MSCs) were obtained from the GMP laboratory of Shanghai East Hospital, Tongji University School of Medicine, Shanghai, China. Bama miniature pigs (Sus scrofa) were purchased from Taihe Biotechnology Co., Ltd. (Taizhou, Jiangsu, China). Antibodies to Cleaved Caspase 3, Phospho-MLKL, CD9, CD81, Phospho- NF-κB p65, Phospho- NF-κB p65, STAT 3, Phospho- STAT 3, BMP 7 and α-Tubulin were purchased from Cell Signaling Technology (Danvers, MA, United States). An antibody to phospho-RIPK3 was purchased from Abcam (Cambridge, United Kingdom). Antibodies to NF-κB p65 and Klotho were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, United States). Antibodies to MLKL, RIPK3, F4/80, VEGFR2 were obtained from Proteintech (Wuhan, China). Antibodies to VEGFA and CD31 were obtained from Invitrogen (Carlsbad, CA, United States). Antibodies to GAPDH and PCNA were obtained from Arigo (Taipei, Taiwan, China). An antibody to NGAL was purchased from Genetex (San Antonio, TX, United States).