Mg132 hy 13259

Anti-MFG-E8 (sc-271574; 1:200 dilution for immunoblotting, immunofluorescence, and immunohistochemistry; 2 µg per 1 mg of total protein for immunoprecipitation), anti-USP14 (sc-398009, 1:200 for immunoblotting, 2 µg per 1 mg of total protein for immunoprecipitation), and anti-GPx4 (sc-166570, 1:400 for immunoblotting) antibodies were purchased from Santa Cruz Biotechnology, USA. Anti-SLC7A11 (12691, 1:1000 for immunoblotting) and anti-Ub (3936, 1:1000) were from Cell Signaling Technology, USA. Anti-Flag (F7425, 1:1000) was from Sigma-Aldrich, USA. Antibodies against β-actin (60008–1-Ig, 1:5000), β-tubulin (10068-1-AP, 1:1000), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (60004-1-Ig, 1:1000) were from Proteintech, China. Anti-USP14 (AF301319, 1:100 for immunofluorescence) was from AiFang Biological, China. Anti-GPx4 (ab125066, Abcam, UK, 1:100), and anti-SLC7A11 (26864-1-AP, Proteintech, China, 1:50) antibodies were used for immunohistochemistry (IHC) staining. Immobilized protein A/G beads (45350) were purchased from Thermo Fisher Scientific, USA. Control IgG (sc-2025) came from Santa Cruz Biotechnology, USA. Cycloheximide (CHX) (S7418), RSL3 (S8155), and Ferrostatin-1 (Fer-1) (S7243) were from Selleck, China. MG132 (HY-13259) was purchased from MedChemExpress, USA.

Antibodies against CDK1 (ab133327), Cyclin B1 (ab32053) and CDC25C (ab32444), Caspase-3 (ab32351), Bcl2 (ab182858), LC3B (ab192890), SQSTM1/P62 (ab109012), and γH2AX (ab81299) were obtained from Abcam (Cambridge, MA, USA). Antibodies against NEK2 (66632-1-lg), TRIM21(12108-1-AP), α-tubulin (11224-1-AP), GAPDH (60004-1-Ig), Bax (60267-1-Ig), Alexa Fluor 594-conjugated donkey anti-rabbit second antibody (SA00013-4), and Alexa Fluor 488-conjugated donkey anti-mouse second antibody (SA00013-1) were obtained from Proteintech (Wuhan, China). An antibody against NEK2 (sc55601) was got from Santa Cruz Biotechnology, Inc (St Louis, MO, USA). The autophagy activator Rapamycin (RAPA, HY-10,219), Cycloheximide (CHX, HY-12,320), and MG132(HY-13,259) were purchased from MedChemExpress (MCE, USA).

BJAB (EBV-negative Burkitt's lymphoma cell line), Raji (EBV-positive Burkitt's lymphoma cell line), B95.8 (EBV-transformed tamarin (Saguinus oedipus) cell line) cells and HONE-1 (human nasopharyngeal carcinoma cell line) were cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS). HEK293 cells were cultured in Dulbecco’s modified Eagle medium supplemented with 10% FBS. All cells were confirmed to be Mycoplasma negative before culture (TaKaRa) and were authenticated by the short tandem repeat Multiamplification Kit (Goldeneye DNA ID system 20A, Peoplespot) every six months. PBMCs were isolated from whole blood of healthy donors by Ficoll centrifugation. Primary B cells were isolated from PBMCs by MojoSort human Pan B cell Isolation Kits (480081, BioLegend) and were cultured in RPMI 1640 medium supplemented with 10% FBS. All cell lines were grown in a humidified incubator at 37 °C with 5% CO₂ and obtained from American Type Culture Collection. CHX (M4879) were purchased from AbMole BioScience. STM2457 (HY-134836) and MG132 (HY-13259) was purchased from MedChemExpress.

The antibodies utilized in this investigation for western blot and immunohistochemistry analysis were as follows: PLAG1 (H00005324-M02, Novus Biologicals, 1:500 for WB, 1:50 for IHC), GPX4 (A5569, Selleck, 1:1000 for WB), AGO2 (#2897, Cell Signaling Technology, 1:1000 for WB), GAPDH (60,004–1-lg, Proteintech, 1:50,000 for WB), 4-HNE (MHN-020P, JalCA, 1:4 for IHC), Ubiquitin(AF1705, Beyotime, 1:1000 for WB). Beyotime and Selleck Chemicals were the sources of the secondary antibody conjugates labeled with Horseradish peroxidase (HRP) and Sorafenib (#S7397), respectively. Cycloheximide (CHX, HY-12320) and MG132 (HY-13259) were purchased from Medchemexpress.

Lipids and detergents (DMPC, DMPG, DMPE, DMPS, POPG, DH⁶PC, Sph and PS (Brain, Porcine)) were from Avanti Polar Lipids. Stable isotopes for NMR spectroscopy experiments were from Cambridge Isotope Laboratories. Anti-human PD-L1 antibodies (ab213524), Alexa488-conjugated anti-IgG (ab150077) and anti IgG-HRP (ab97051) were from Abcam. Anti-V5 antibodies (96025) were from Thermo and FITC anti PD-L1 antibodies (393606) were from Biolegend. MG132 (HY-13259) and metformin (HY-B0627) were from MedChem Express (MCE). The HEK293T cell line was a generous gift from Liming Sun (SIBCB). The RKO cell lines for expressing PD-L1 were generous gifts from Jie Xu (Fudan University), other cell lines used in the paper were from Cell Bank of Chinese Academy of Sciences.

Tim-AIII (purity >98%) was purchased from Chengdu Herbpurify Co., LTD (Z-019, China) and was dissolved in dimethyl sulfoxide (DMSO, 196055, MP Biomedicals, USA) to prepare a stock solution of 10 mM and stored at -20°C. Mouse anti-GPX4 (67763-1-Ig) antibody, rabbit anti-HSP90 (13171-1-AP), E-Cadherin (20874-1-AP), Vimentin (10366-1-AP), Snail-1 (13099-1-AP), Snail-2 (12129-1-AP), N-Cadherin (22018-1-AP) antibodies and HRP-conjugated anti-heavy chain of rabbit IgG antibody (SA00001-7H) were purchased from Proteintech (Wuhan, China). Rabbit anti-GPX4 (A1933), FTL (A11241), HMOX-1 (A1346), SLC40A1 (A14885), SLC7A11 (A13685) Ubiquitin (A19686) and β-actin (AC004) antibodies were purchased from ABclonal (Wuhan, China). The HRP-labeled goat anti-rabbit IgG and HRP-labeled goat anti-mouse secondary antibody was purchased from Beyotime (Shanghai, China). Chloroquine (CQ, HY-17589A), Z-VAD-FMK (HY-16658B), Necrostatin-1 (Nec-1, HY-15760), Ferrostatin-1 (Fer-1, HY-100579), N-acetylcystein (NAC, HY-B0215), tanespimycin (HY-10211), trolox (HY-101445), cycloheximide (CHX, HY-12320) and MG-132 (HY-13259) were purchased from Med Chem Express (Shanghai, China). The recombinant human HSP90 alpha protein (ab48801) was provided by Abcam (Shanghai, China). The recombinant Human GPX4 protein (P0633) was provided by Fine Biotech (Wuhan, China).

The cell lines, used in this study, were acquired from the Chinese Academy of Sciences (Shanghai, China). The human BCa cell lines namely, RT4, T24, UMUC3, EJ, and SW780 were used. The immortalized human normal bladder epithelial cell line, SV-HUC-1 was also used. Here, the RT4 cell line was cultured in the McCoy's 5A medium (Thermo Fisher Scientific, Inc. USA), SV-HUC-1 in the F12k medium (Sigma-Aldrich, St. Louis, MO, USA), and T24, UMUC3, EJ, and SW780 cell lines were cultured in the RPMI-1640 medium (Thermo Fisher Scientific, Inc. USA). All cell culture mediums were supplemented with 10% fetal calf serum (FCS; Thermo Fisher Scientific, Inc. USA) and 1% penicillin /streptomycin (Hyclone, Logan, UT, USA). These cell lines were incubated in an incubator containing 5% CO₂ at 37°C. Other compounds like chloroquine (CQ, HY-17589A), necrostatin 1 (nec-1, HY-15760), erastin (HY-15763), ferrostatin-1 (fer-1, HY-100579), RSL3 (HY-100218A), NCT-502 (HY-117240), actinomycin D (HY-17559), and MG-132 (HY-13259) were purchased from MedChemExpress (Shanghai, China).