Hrp labeled anti mouse secondary antibody

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.

Purified proteins (5 μg) were mixed with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer with or without 20 mM dithiothreitol (DTT) and denatured at 100°C for 5 min. SDS-PAGE was performed using 4% stacking gels at 80 V for 15 min and 15% separating gels at 180 V for approximately 50 min (Mini-PROTEAN^® Tetra System, Bio-Rad, Hercules, CA, USA). After electrophoresis, the gels were stained with Coomassie Blue R250.
For western blotting, 0.1–0.5 μg proteins were separated as described above and transferred onto PVDF membranes (Immobilon-P, Millipore, Darmstadt, Germany) at 300 mA for 1 h (Mini-PROTEAN^® Tetra System; Bio-Rad). The membranes were then blocked with 5% non-fat milk in Tris-buffered saline with 0.05% Tween-20 (TBST, pH 7.6) for 1 h at room temperature, and PDGF-B was detected by incubation with a mouse anti-human PDGF-B antibody (1:1,000; sc-365805, Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 h at room temperature. After washing with TBST three times, the membranes were incubated with an HRP-labeled anti-mouse secondary antibody (1:10,000; #7076, Cell Signaling Technology, Danvers, MA, USA) for 1 h at room temperature. Target proteins were visualized using a LAS4000 mini gel imaging system (GE Healthcare, Fairfield, CT, USA) after adding an HRP substrate (Immobilon; Millipore).

Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), bovine calf serum (BS), penicillin/streptomycin, and trypsin 0.25% were purchased from Hyclone (GE Healthcare Life Science, Pittsburgh, PA, United States). The radio-labeled [³H]-mitoxantrone (4 Ci/mmol) was purchased from Moravek Biochem-icals, Inc. (Brea, CA, United States). Phosphate buffered saline (PBS) and dimethyl sulfoxide (DMSO) were obtained from Thermo Fisher Scientific Inc. (Rockford, IL, United States). Mitoxantrone (MX), SN-38, cisplatin, geneticin (G418), and Ko143 were obtained from Enzo Life Sciences (Farmingdale, NY, United States). CBZ was purchased from Chemietek Company (Indianapolis, IL, United States). The mouse monoclonal antibodies for ABCG2 and glyceraldehyde phosphate dehydrogenase (GAPDH) were obtained from Thermo Fisher Scientific Inc. (Rockford, IL, United States). The rabbit monoclonal antibodies against human DNA topoisomerase I, the HRP-labeled anti-mouse secondary antibody, and the HRP-linked anti-rabbit secondary antibody were purchased from Cell Signaling Technology (Danvers, MA, United States). TPT, 3-(4, 5-dimethylthiazol-yl)-2, 5-diphenyltetrazolium bromide (MTT), and all other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, United States).