Anti mouse 7076

M-PER mammalian protein extraction reagent (Thermo Fisher), containing a protease inhibitor cocktail, was used to extract protein lysate from transfected cells (Thermo Fisher). SDS-PAGE was used to separate 40 μg of protein lysate, followed by blotting onto a nitrocellulose membrane (Bio-Rad, USA). The membrane was blocked using 5% skimmed milk in Tris-buffered saline containing 0.1% Tween 20 (TBST) for 1 h. Subsequently, blot was incubated overnight at 4 °C with primary antibodies against GLUT2 (Anti-rabbit; 1:1000, #ab54460, Abcam, UK), INSRβ (Antimouse; 1:1000; #ab69508, Abcam, UK), INSRα (Anti-rabbit; 1:1000; #ab5500, Abcam, UK), Insulin (Anti-mouse; 1:1000; #8138s, Cell signaling Technology, USA), GCK (Anti-rabbit; 1:500; #ab37796, Abcam, UK) PDX1 (Anti-rabbit; 1:3000, #ab47267, Abcam, UK), or β-actin (1:5000; #A5441, Sigma-Aldrich, Germany). The membrane was incubated with secondary antibodies (anti-mouse #7076S and antirabbit #7074S, Cell signaling Technology, USA) at 1:1000 dilutions at room temperature for 1 h. ECL substrate kit (Bio-Rad, USA) was used to detect chemiluminescence. Bio-Rad Image Lab software (Bio-Rad, USA) was used to detect protein bands. Quantification of the bands was done using Image J software. In all experiments, β-actin was used as an endogenous control (see Supplementary Information).

After 24 h, cells were harvested and dissolved in lysis buffer (100 mM Tris-HCl, pH 6.8, 4% SDS, 20% glycerin, and 200 mM dithiothreitol). Protein concentration was measured using the Pierce BCA Protein Assay Kit (Thermo Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Samples were mixed with Laemmle’s loading buffer and boiled for 7 min. Equal amounts (20–40 µg) of protein extracts were loaded to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE, 12% gel) at 100 mV, followed by blotting onto nitrocellulose membranes for 1 h 50 min at 110 V on ice. After transfer, nitrocellulose membranes stained with Ponceau S. Membranes were blocked for 1 h with 5% nonfat milk (AppliChemGmbH, Darmstadt, Germany, A0830) in tris-buffered saline (TBS) at room temperature, probed overnight with the appropriate primary antibodies (1:3000–1:5000) and followed by incubation with horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology, anti-mouse 7076S or anti-rabbit 7074S, 1:4000) for 1 h with 2.5% nonfat milk in Tris-buffered saline (TBS) at room temperature. Detection was visualized with ECL (Amersham Biosciences, Piscataway, NJ, USA) and ChemiDoc MP Imaging System (BioRad). The quantitative analysis of Western blot data was accomplished using ImageJ Software (NIH, Bethesda, Rockville, MD, USA).

Tunicamycin (Tuni, #sc-3506) and JC-1 (#sc-364116A) were purchased from Santa Cruz Biotechnology. RIPA Buffer (#R0278), Protease Inhibitor Cocktail (PIC, #P8340), Propidium Iodide (PI, #P4864) were purchased from Sigma; 3- [4, 5-dimethylthiazol-2-yl]− 2,5-di-phenyltetrazolium bromide (MTT, #33611) was obtained from SRL; beta-cyclodextrin (β-CD, #C0900), and 3-(2-Benzothiazolyl)− 7-(diethylamino), coumarin (C6, #B2088) were purchased from TCI Chemicals. MitoSOX (#M36008), Fura-2 AM cell permeant(#F1221), ER Tracker Green (#E34251) was purchased from Invitrogen. FITC conjugated AnnexinV (#A13199), AnnexinV binding buffer (#V13246), Enhanced Chemiluminescence (ECL, #32106) and Antifademountant (4′-6-diamidino-2-phenylindole, #P36962) were procured from Thermo Fisher Scientific. Antibodies (TOM20 #D8T4N, DRP1 #D6C7, phospho-DRP1 #D9A1, IRE1α #14C10, BiP #C50B12, Calnexin #C5C9) were obtained from Cell Signaling Technology (CST, USA) and Santa Cruz Biotechnology (β-actin #sc-69879, GAPDH #sc-365062) and secondary antibodies- (anti-mouse #7076 S and anti-rabbit #7074P2) were procured from Cell Signaling Technology.

The dual Src/Abl inhibitor, bosutinib, was purchased from LC Laboratory (B-1788, Woburn, MA, USA). Doxorubicin (dox, D1515), etoposide (VP-16, E1383), and anti-β-Actin antibody (A2228) were purchased from Sigma (Sigma-Aldrich Corp, St. Louis, MO, USA). The antibodies against p-Src (Y416, 6943S), Src (2108S), p-c-Abl (Y245, 2868S), c-Abl (sc-56887), p-STAT3 (Y705, 4904S), STAT3 (4904S), p-ERK (T202/Y204, 9106L), ERK (4695S), p-S6 (Ser235/236) ribosomal protein (4858S), S6 ribosomal protein (2217S), Caspase 3 (9662S), PARP (9532S), and anti-Mouse (7076S) or anti-Rabbit (7074S) IgG were purchased from Cell Signaling Technology (Danvers, MA, USA).

Lysate from activated and non-activated spleen T cells, activated spleen B cells or fibroblasts were separated on 8% SDS-PAGE gel. After transfer to nitrocellulose membranes, the following primary antibodies were used: rabbit monoclonal anti-CTPS1 EPR8086 (Abcam ab133743) or rabbit polyclonal anti-CTPS2 (C-ter) (Abcam ab190462) (dilution 1/1000) and mouse monoclonal anti-actin (Santa-Cruz-4778) (dilution 1/5000). Membranes were then washed and incubated with anti-mouse (7076S) or anti-rabbit HRP (7074S) conjugated antibodies (dilution 1/10000) from Cell Signalling Technology and Pierce ECL western blotting substrate was used for detection. Uncropped blots are supplied in the Source Data File.

TAK1 inhibitor NG25 (HY-15434) was purchased from MedChem Express (Monmouth Junction, NJ, USA) and was prepared according to the manufacturer’s recommendations. The antibodies against PARP (9532), Caspase 3 (9662), Caspase 7 (12827), IκBα (9242), phospho-p38 MAP kinase (Thr180/Tyr182) (9211), p38 MAP kinase (8690), anti-Mouse (7076S) and anti-Rabbit (7074S) IgG were purchased from Cell Signaling Technology (Danvers, MA, USA). Doxorubicin (Dox, D1515) and antibody against β-actin (A2228) were obtained from Sigma-Aldrich Corp (St. Louis, MO, USA).

TMT, ascorbic acid, dimethyl sulfoxide (DMSO), 2′,7′-dichlorofluorescein diacetate (DCF-DA), 2′,3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), lactate dehydrogenase (LDH) assay kit, superoxide dismutase (SOD) assay kit, and all other chemicals were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Phosphoprotein kinase B (p-Akt; Ser 473; 9271S), BAX (2772S), and anti-rabbit (7074S) and anti-mouse (7076S) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Phospho-c-Jun N-terminal kinases (p-JNK; sc-6254), cytochrome c (sc-13560), p-tau (Ser 404; sc-12952), and β-actin (sc-69879) antibodies were obtained from Santa Cruz Biotechnology (Dallas, TX, USA).