Ab184718

Protein concentrations were measured using the Micro BCA™ Protein Assay Kit (23235; Thermo Fisher) and proteins were resolved by 10% or 14% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane (Amersham Pharmacia, Uppsala, Sweden). The levels of HIF-1α (ab179483; Abcam), signal transducer and activator of transcription 3 (STAT3; #9139; Cell Signaling Technology, Danvers, MA, USA), p-STAT3^Tyr705 (#9145; Cell Signaling Technology), p-STAT3^Ser727 (#9136; Cell Signaling Technology), lysosomal-associated membrane protein 1 (LAMP1; sc-20011; Santa Cruz Biotechnology, Santa Cruz, TX, USA), p62 (ab56416; Abcam), microtubule-associated protein 1 light chain 3 (LC3; ab48394; Abcam), p-MLKL (ab184718; Abcam), MLKL (Abcam), and GAPDH (ab181602; Abcam) were detected by Western blotting. Signals were detected using anti-mouse or anti-rabbit HRP-conjugated secondary antibodies. The western blots were quantification using Fiji/imageJ program. Target protein was normalized with GAPDH and, phospho-proteins normalized with total protein and GAPDH.

The antibodies used for immunoblotting included: mouse monoclonal antibody against GAPDH (RM2002, Beijing Ray, Beijing, China); rabbit monoclonal antibodies against HtrA2 (ab75982, Abcam, Cambridge, MA, USA), p-RIPK1 (65746, Danvers, MA, CST, USA), RIPK1 (3493, CST), p-RIPK3 (93654, CST), human p-MLKL (91689, CST), human MLKL (ab184718, Abcam), and mouse p-MLKL (ab196436, Abcam); rabbit polyclonal antibodies against RIPK3 (ab56164, Abcam) and mouse MLKL (ab172868, Abcam); and goat anti-mouse (R3001, Beijing Ray) or goat anti-rabbit (R3002, Beijing Ray) HRP-conjugated secondary antibody.
The antibodies used for IHC staining included: CD11b (ab133357, Abcam), S100a9 (73425, CST, USA), HtrA2 (ab75982, Abcam), MPO (ab9535, Abcam), and F4/80 (ab111101, Abcam).
Other reagents included: DSS (36,000–50,000 kD, MP Biomedicals, Santa Ana, CA, USA), UCF-101 (Cayman Chemical, USA), Nec-1s, BV-6, Z-VAD (Selleck, Houston, TX, USA), mouse TNF-α (R&D, Minneapolis, MN, USA), Cell Counting Kit-8 (CCK-8, MedChemExpress, Monmouth Junction, NJ, USA), FITC-dextran (4 kDa, Sigma, St. Louis, MO, USA).

The experimental procedures were performed as previously reported [2 (link)]. The extraction of total protein was performed on ice throughout the entire process. The protein concentration was determined by Bradford method. Total proteins were separated on a 10% SDS-PAGE gel, then transferred onto PVDF membranes (Bio-Rad Laboratories Inc., CA, USA). After blocked with 5% non-fat milk for 1 h, membranes were incubated with antibodies RIP3 (ab62344, Abcam, Cambridge, UK), MLKL (ab184718, Abcam), MyD88 (ab219413, Abcam), TLR4 (ab13556, Abcam), p-IκB (CY6280, Abways Technology Inc., Shanghai, China), IκB (CY2327, Abways) and p65 (ab32536, Abcam). β-actin (AB0011, Abways) or histone H3 (ab1791, Abcam) was used as the loading control. Then, membranes were incubated with Peroxidase AffiniPure Goat Anti-Rabbit IgG (H + L) (111-035-003, 1:10,000, Jackson ImmunoResearch Inc., PA, USA) for 1 h and then HRP chemiluminescence detection reagent (Tiangen Biotech, Beijing, China). Tanon 5200 automatic chemiluminescence image analysis system (Tanon, Beijing, China) was applied for ECL imaging. Image J software (Rawak Software Inc., Stuttgart, Germany) performed semi-quantitative analysis.

The 3-acyl isoquinolin-1(2H)-one complex 4f was synthesized according to a known procedure developed in our previous work. Cell counting kits-8 (CCK8) was purchased from Beyotime Biotechnology, dimethyl sulfoxide (DMSO) and RIPA lysis buffer were purchased from Sigma (Beverly, MA, USA). Primary antibodies such as anti-caspase3 antibody (cat#14220T), anti-cleaved-caspase9 antibody (cat#52873), anti-cleaved-caspase7 antibody (cat#8438), anti-parp antibody (cat#9542), anti-bcl-2 antibody (cat#15071T), anti-bax antibody (cat#5023), anti-β-tubulin antibody (cat#2128s), anti-ERK1/2 antibody (cat#4695T), anti-p-ERK1/2 antibody (cat#4370T), anti-MEK1/2 antibody (cat#9126s), anti-p-MEK1/2 antibody (cat#9154T), (HRP)-labeled anti-rabbit secondary antibody (Cat#7074) and (HRP)-labeled anti-mouse secondary antibody (Cat#7076) were purchased from Cell Signaling Technology. Anti-GSDME antibody (ab215191), anti-GSDMD antibody (ab210070), anti-p-MLKL antibody (ab187091) and anti-MLKL antibody (ab184718) were purchased from Abcam. Anti-CDK1 antibody (AF1516) was purchased from Beyotime Biotechnology.

Histones were extracted using a histone extraction kit (Abcam Cambridge, MA) according to manufacturer protocol. To induce necroptosis and serve as a positive control, cells were treated with pan-caspase inhibitor Q-VD-OPh (25 μM) and BV6 (7.5 μM) for 30 min followed by incubation with TNF-α (50 ng/ml). Western blot was performed using methods previously described^{16 (link)}. Antibodies against p27 (3686), RIP1 (4926), acetylated H3K9 (9649S), and H3K14 (7627S) were obtained from Cell Signaling Technology (Danvers, MA). Antibodies against phosphorylated MLKL (ab187091), MLKL (ab184718), and RIP3 (ab56164) were obtained from Abcam (Cambridge, MA). Antibody against H3 (06–755) was purchased from Upstate (Lake Placid, NY) and tubulin (CP-06) from Calbiochem (San Diego, CA). HRP conjugated secondary antibodies were obtained from Sigma Aldrich (St. Louis, MO).

The extraction of protein was performed as described^{76 (link)}. Proteins were separated by 12% Bis-Tris Plus gels (Genscript, China), transferred to PVDF membrane (Millipore, USA) by 350 mA for 90 min, and blocked for 1 hour at room temperature. Primary antibodies used to probe blots were mouse anti-TICAM-1 (sc-514384, mAb, Santa, USA), mouse anti-FLIPS/L (sc-5276, mAb, Santa, USA), rabbit anti-cleaved caspase-8 (9496, mAb, CST, USA), rabbit anti-total caspase-8 (ab108333, mAb, Abcam, USA), rabbit anti-RIPK1/RIP1 (NBP1-77077, polyclonal Ab, Novus, USA), rabbit anti-RIP3 (ab152130, polyclonal Ab, Abcam, USA), rabbit anti-MLKL (ab184718, mAb, Abcam, USA), rabbit anti-p-MLKL (phospho S358) (ab187091, mAb, Abcam, USA), rabbit anti-TOP1 (20705-1-AP, polyclonal Ab, Proteintech, USA), rabbit anti-BET (13232, mAb, CST, USA), rabbit anti-CDK9 (2316, mAb, CST, USA) and rabbit anti-GAPDH (MB001, mAb, Bioworld, USA), which were incubated overnight at 4 °C. Subsequently, HRP-conjugated secondary antibodies including anti-rabbit and anti-mouse antibodies (Fcmacs, China) were incubated for 1 hour. Blots were then visualized by a chemiluminescent imaging system (Tanon, Shanghai, China).

Cell lysates were heat denatured, separated by SDS‒PAGE, transferred to PVDF membranes (abs931, Absin, Shanghai, China), and blocked with 5% skimmed milk. Then, membranes were incubated, respectively with anti-BRAF (1:1000, ab33899), anti-GPX4 (1:1000, ab125066), anti-PUMA (1:1000, ab9643), anti-LC3B (1:2000, ab192890), anti-MLKL (1:500, ab184718), anti-phospho-MLKL (1:1000, ab196436), anti-ERK1/2 (1:1000, ab184699), anti-Ubiquitin (1:1000, ab134953), anti-AMPKα (1:2000, ab32047), anti-p62 (1:1000, ab109012), anti-Beclin1 (1:2000, ab207612) primary antibodies from Abcam (UK), anti-Alpha Tubulin (1:1000, 11224-1-AP), anti-FOXO3a (1:1000, 10849-1-AP), anti- SLC7A11 (1:1000, 26864-1-AP), ati-H3 (1:1000, 17168-1-AP) primary antibodies from Proteintech (IL, USA), anti-Cleaved Caspase-3 (1:1000, #9661), anti-phospho-Beclin1 (1:1000, #14717), anti-phosphor-AMPKα (1:1000, #2535), anti-phospho-FOXO3a (1:1000, #64616), anti-phospho-ERK1/2 (1:1000, #4370) primary antibodies from Cell Signaling Technology (MA, USA) and anti-ATG5 (1:1000, ET1611-38) from HUABIO (Hangzhou, China) overnight at 4 °C and anti-rabbit (1:5000, ab205718, absin, Shanghai, China) or anti-mouse (1:5000, abs20039, absin, Shanghai, China) secondary antibodies for 1 h at room temperature. Immunoblots were visualized using an ECL detection reagent (BMU101-CN, Abbkine Scientific, Wuhan, China).