Preparation of total cell lysates and Western blot analysis were performed as described previously [48 (link)]. Protein content was detected using Bradford method. SDS-PAGE electrophoresis, transfer, immunostaining, and ECL detection were carried out according to standard protocols of Bio-Rad Laboratories and antibody manufacturers. Primary antibodies against the following proteins were utilized: IGFBP3 (B-5, 1:500, from Santa Cruz Biotech.), phospho-p70S6 kinase (Thr389, 1:1000), phospho-S6 (Ser240/244, 1:1000), phospho-4E-BP1 (Thr37/46, 1:1000), phospho-ERK1/2 (Thr202/Tyr204, 1:2000), ERK2 (1:3000), phospho-Akt (Thr308, 1:700), Akt (1:1000), and GAPDH (clone 14C10, 1:4000) – all from Cell Signaling Technology. Secondary antibodies for immunoblotting – GAR-HRP (1:10000) and GAM-HRP (1:10000) were also from Cell Signaling Technology.
Gapdh clone 14c10
Manufactured by Cell Signaling Technology
Sourced in United States
GAPDH (clone 14C10) is a primary antibody produced by Cell Signaling Technology. It targets the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein, which is a key enzyme involved in the glycolytic pathway.
Automatically generated - may contain errors
Lab products found in correlation
Gar hrp, by Cell Signaling Technology (6 mentions)
Gam hrp, by Cell Signaling Technology (5 mentions)
Enhanced chemi luminescence (ecl), by Thermo Fisher Scientific (5 mentions)
Phospho p53 ser15 clone 16g8, by Cell Signaling Technology (3 mentions)
Phospho rb ser807 811, by Cell Signaling Technology (3 mentions)
Etoposide, by Enzo Life Sciences (2 mentions)
Nigericin, by Cayman Chemical (2 mentions)
P21waf1 cip1 clone 12d1, by Cell Signaling Technology (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Progesterone receptor a b clone d8q2j, by Cell Signaling Technology (2 mentions)
E cadherin clone hecd 1, by Abcam (2 mentions)
Phospho akt, by Cell Signaling Technology (1 mentions)
Phospho 4e bp1 thr37 46, by Cell Signaling Technology (1 mentions)
Phospho erk1 2 thr202 tyr204, by Cell Signaling Technology (1 mentions)
Phospho s6 ser240 244, by Cell Signaling Technology (1 mentions)
Igfbp3 b 5, by Santa Cruz Biotechnology (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
Sitagliptin, by Merck Group (1 mentions)
Vildagliptin, by Cayman Chemical (1 mentions)
Saxagliptin, by LGC (1 mentions)
22 protocols using gapdh clone 14c10
1
Immunoblotting Analysis of Signaling Pathways
2
Caspase Activation and Pyroptosis Assays
3
Synthesis and Characterization of Compound 8j
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Compound 8j was synthesized according to previously published protocols29 (link). VbP (Talabostat mesylate) was purchased from R&D Systems and was resuspended in DMSO containing 0.1% TFA to prevent cyclization. Bortezomib was purchased from LC laboratories, zVAD-FMK from Ubpbio, VX-765 from Apexbio Technology LLC, and etoposide from Enzo Life Sciences. LPS (from E. coli O111:B4) was purchased from Invivogen, Nigericin from Cayman Chemicals, Bestatin Methyl Ester from Santa Cruz, and MCC950 from AdipoGen. Antibodies used were: hCASP1 (no. 2225, Cell Signaling Technology), GAPDH (clone 14C10, Cell Signaling Technology), hGSDMD (NBP2-3342, Novus Biologicals), PARP (no. 9542, Cell Signaling Technology), hNLRP1 (AF6788, R&D Systems), hCARD8 (no. ab24186, Abcam), mGSDMD (no. ab209845, Abcam), GSDMD (no. 219800, Abcam), α-Actin (no. A4700, Sigma-Aldrich), mCD45 (no. 103108, Biolegend, FITC conjugate, clone 30-F11), mCD3 (no. 100235, Biolegend, APC conjugate, clone 17A2), rCD3 (no. 201411, Biolegend, PE conjugate, clone 1F4), rCD6 (no. 554904, BD Biosciences, FITC conjugate, clone OX-52), mCaspase-1 (AG20B-0042, Adipogen), mIL-1β (no. D4T2D, Cell Signaling Technologies), NLRC4 (no. ab201792, Abcam), and NLRP3 (no. ab210491, Abcam).
4
Western Blotting: Protein Expression Analysis
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Western blotting was performed as described previously (Borodkina et al., 2014 (link)). SDS-PAGE electrophoresis, transfer to nitrocellulose membrane and immunoblotting with ECL (Thermo Fisher Scientific, United States) detection were performed according to standard manufacturer’s protocols (Bio-Rad Laboratories, United States). Antibodies against the following proteins were used: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (clone 14C10) (1:1000, #2118, Cell Signaling Technology, United States), phospho-p53 (Ser15) (clone 16G8) (1:700, #9286, Cell Signaling Technology, United States), p21Waf1/Cip1 (clone 12D1) (1:1000, #2947, Cell Signaling Technology, United States), phospho-Rb (Ser 807/811) (1:1000, #8516, Cell Signaling Technology, United States), well as horseradish peroxidase-conjugated goat anti-rabbit IG (GAR-HRP, Cell Signaling Technology, United States) (1:10000).
5
Comprehensive Western Blotting Protocol
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Western blotting was performed as described previously [22 (link)]. SDS-PAGE electrophoresis, transfer to nitrocellulose membrane and immunoblotting with ECL (Thermo Scientific, USA) detection were performed according to standard manufacturer’s protocols (Bio-Rad Laboratories, USA). Antibodies against the following proteins were used: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (clone 14C10) (1:1000, #2118, Cell Signaling, USA), phospho-p53 (Ser15) (clone 16G8) (1:700, #9286, Cell Signaling, USA), p21Waf1/Cip1 (clone 12D1) (1:1000, #2947, Cell Signaling, USA), phospho-Rb (Ser807/811) (1:1000, #8516, Cell Signaling, USA), phospho-ATM (Ser1981) (clone D6H9) (1:1000, #5883, Cell Signaling, MA, USA), phospho-Histone H2A.X (Ser139) (clone JBW301) (1:1000, #05-636, Merck Millipore, Germany), CD63 (1:500, ab68418, Abcam, UK), HSP70 (clone 2H9) (#MABE1130, Merck Millipore, Germany), PAI-1 (D9C4) (1:1000, #11907, Cell Signaling, USA) as well as horseradish peroxidase-conjugated goat anti-rabbit IG (GAR-HRP, Cell Signaling, USA) (1:10000) and antimouse IG (GAM-HRP, Cell Signaling, USA) (1:10000). Full size blots are provided in the Supplementary Figure 1 .
6
Antibody Validation for UPR Signaling
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Polyclonal antibodies for phosphorylated eIF2α (Ser51) and TBP (TATA-binding protein), as well as monoclonal antibodies for β-actin (clone 13E5) and GAPDH (clone 14C10), were purchased from Cell Signaling Technology (Danvers, MA, USA). Polyclonal antibodies for ATF6α and ATF4 (CREB2) were from Santa Cruz Biotechnology (Dallas, TX, USA). The antibody for ATF3 was purchased from GeneTex (Irvine, CA, USA), and the antibody for phosphorylated IRE1α (Ser724) was purchased from Thermo Fisher Scientific (Waltham, MA, USA). 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), N-(2-hydroxyethyl)-piperazine-N′-3-propanesulfonic acid (EPPS), 2-(4-morpholino)-ethanesulfonic acid (MES), and the protease inhibitor cocktail were from Sigma-Aldrich (St Louis, MO, USA) and Thermo Fisher Scientific (Waltham, MA, USA). The GPR4 inhibitor, 2-Ethyl-3-(4-((E)-3-(4-isopropyl-piperazin-1-yl)-propenyl)-benzyl)-5,7-dimethyl-3H-imidazo(4,5-b)pyridine, was purchased from Dalton Pharma Services (Toronto, ON, Canada).
7
Quantification of Glut9 Protein Expression
8
Immunoblotting Analysis of Cell Signaling
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Immunoblotting analysis was performed as described previously [11 (link)]. SDS-PAGE electrophoresis, transfer to nitrocellulose membrane and immunoblotting with ECL (Thermo Scientific, CA, USA) detection were performed according to standard manufacturer’s protocols (Bio-Rad Laboratories, USA). Antibodies against the following proteins were used: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (clone 14C10) (1:1000, #2118, Cell Signaling, USA), phospho-Rb (Ser807/811) (1:1000, #8516, Cell Signaling, USA), phospho-p53 (Ser15) (clone 16G8) (1:700, #9286, Cell Signaling, USA), p21Waf1/Cip1 (clone 12D1) (1:1000, #2947, Cell Signaling, USA), phospho-ATM (Ser1981) (clone D6H9) (1:1000, #5883, Cell Signaling, USA), phospo-p38 (Thr180/Tyr182) (1:1000, #9211, Cell Signaling, USA) as well as horseradish peroxidase-conjugated goat anti-rabbit IG (1:10000, #7074, GAR-HRP, Cell Signaling, USA) () and antimouse IG (1:10000, #7076, GAM-HRP, Cell Signaling, USA) (). Hyperfilm (CEA) was from Amersham (Sweden). Equal protein loading was confirmed by Ponceau S (Sigma-Aldrich, USA) staining.
9
Comprehensive Protein and mRNA Analysis
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Western blot was performed as previously described14 (link). Antibodies used: HA-Tag (Cell Signalling #3724; dilution 1:10,000); MITF (Thermo Fisher Scientific MA5-14146; dilution 1:1000); β-Actin (Cell Signalling #3700; dilution 1:2000); GAPDH (clone 14C10; Cell Signalling #2218; dilution 1:1000); CRYAB (Cell Signalling #45844s; dilution 1:1000).
RNA was extracted using NucleoSpin® RNA isolation kit from Macherey-Nagel (ref: 740955.240C). For patients and animal tissues a Trizol-based implementation of the NucleoSpin® RNA isolation kit protocol was used as reported14 (link). One microgram of total RNA was used for cDNA synthesis using MaximaTM H Minus cDNA Synthesis Master Mix (Invitrogen M1682). Quantitative Real-Time PCR (qRTPCR) was performed as previously described14 (link). Universal Probe Library (Roche) primers and probes employed are detailed in Supplementary Table5 . GAPDH (Hs02758991_g1) housekeeping assay from Applied Biosystems was used for data normalization.
For transcriptomics analysis in PC3 TRIPZ-HA-Flag-MITFA cells, Illumina whole genome -HumanHT-12_V4.0 (DirHyb, nt) method was used as reported14 (link). A hypergeometric test was used to detect enriched dataset categories.
RNA was extracted using NucleoSpin® RNA isolation kit from Macherey-Nagel (ref: 740955.240C). For patients and animal tissues a Trizol-based implementation of the NucleoSpin® RNA isolation kit protocol was used as reported14 (link). One microgram of total RNA was used for cDNA synthesis using MaximaTM H Minus cDNA Synthesis Master Mix (Invitrogen M1682). Quantitative Real-Time PCR (qRTPCR) was performed as previously described14 (link). Universal Probe Library (Roche) primers and probes employed are detailed in Supplementary Table
For transcriptomics analysis in PC3 TRIPZ-HA-Flag-MITFA cells, Illumina whole genome -HumanHT-12_V4.0 (DirHyb, nt) method was used as reported14 (link). A hypergeometric test was used to detect enriched dataset categories.
10
Western Blot Analysis of T-PLL Cells
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We performed Western blots of whole-cell lysates of primary T-PLL cells and CD3+ pan T-cell isolated from age matched healthy controls by magnetic-bead based cell enrichment. Positive selection of CD3+ pan T-cells of healthy controls was performed according to the manufactures instruction (Biolegend). Following antibodies from Cell Signaling Technology were used in a 1:1000 dilution according to the manufacturer’s instruction: phospho-STAT5TYR694 (clone C11C5, catalogue #9359), STAT5 (polyclonal, #9363), and GAPDH (clone 14C10, #2118). As a secondary antibody we made use of HRP-coupled anti-rabbit (Dianova) in a 1:5000 dilution. Immunoblots were done using Western Bright ECL (Advansta, Menlo Park, CA, USA) and luminescence intensities were evaluated by densitometry (ImageJ software, National Institute of Health, Bethesda, MD, USA). Uncropped images of the immunoblots are displayed in Figure S5 .
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