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3 protocols using jtc 801

1

Comprehensive Immunoblot Analysis

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The antibodies to CA9, p-RelA, RelA, p-IKKβ, IKKβ, p-IκBα, IκBα, LDH, HIF1α, SQSTM1, cleaved-caspase-3, cleaved-PARP1, NRF2, and actin were obtained from Cell Signaling Technology. The antibodies to GPX4 and CA9 were obtained from Abcam. The antibodies to LC3, MLKL, RIPK3, OPRL1, OPRM1, and HMGB1 were obtained from NOVUS. Desferrioxamine, β-mercaptoethanol, N-acetyl-l-cysteine, N-Acetyl-l-alaine, hydrogen peroxide solution, gemcitabine hydrochloride, sulfasalazine, cobalt chloride, cycloheximide, tocopherol, necrosulfonamide, hydroxychloroquine sulfate, EDTA, cytochalasin B, cytochalasin D, paclitaxel, crystal violet, protease inhibitor cocktail, Z-VAD-FMK, TNF, staurosporine, cycloheximide, and lipopolysaccharides were obtained from Sigma-Aldrich. Erastin, ferrostatin-1, lapatinib, JTC801, and compound libraries were obtained from Selleck Chemicals. BANORL24, SB612111, UFP101, and Trap101 were obtained from Tocris Bioscience.
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Compound Library Screening for Bioactive Inhibitors

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An 1,833-member bioactive compound library, and an independent 86-member PI3K signaling inhibitor library comprising mTOR, PI3K and AKT pathway inhibitors, were obtained from Selleck Chemicals and stored at −80°C. Erastin2, annotated as compound 35MEW28 (Ref.51 (link)) and ML162 were synthesized by Acme Bioscience. Erastin was the kind gift of B. Stockwell (Columbia). Chemical used (supplier) were: DMSO, ferrostatin-1, thapsigargin, tunicamycin, cycloheximide, L-arginine, D-arginine and L-citrulline (Sigma-Aldrich Corporation); bortezomib, rapamycin, etoposide and buthionine sulfoximine (Thermo-Fisher Scientific); INK 128, AZD8055, vinblastine, camptothecin, sorafenib, bazedoxifene, raloxifene and JTC-801 (Selleck Chemicals); and GCN2iB (MedChemExpress). buthionine sulfoximine was dissolved directly into cell media. All other drugs were prepared as stock solutions in DMSO. Stock solutions were stored at −20˚C.
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3

Compound Library Screening for Bioactive Inhibitors

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An 1,833-member bioactive compound library, and an independent 86-member PI3K signaling inhibitor library comprising mTOR, PI3K and AKT pathway inhibitors, were obtained from Selleck Chemicals and stored at −80°C. Erastin2, annotated as compound 35MEW28 (Ref.51 (link)) and ML162 were synthesized by Acme Bioscience. Erastin was the kind gift of B. Stockwell (Columbia). Chemical used (supplier) were: DMSO, ferrostatin-1, thapsigargin, tunicamycin, cycloheximide, L-arginine, D-arginine and L-citrulline (Sigma-Aldrich Corporation); bortezomib, rapamycin, etoposide and buthionine sulfoximine (Thermo-Fisher Scientific); INK 128, AZD8055, vinblastine, camptothecin, sorafenib, bazedoxifene, raloxifene and JTC-801 (Selleck Chemicals); and GCN2iB (MedChemExpress). buthionine sulfoximine was dissolved directly into cell media. All other drugs were prepared as stock solutions in DMSO. Stock solutions were stored at −20˚C.
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