Cells were lysed in mammalian cell lysis buffer (50mM Tris pH 7.5, 150mM NaCl and 0.5% NP40) containing a protease and phosphatase inhibitor cocktail (Bimake.com ) and 1 μM MG132 (Selleckchem). To generate PDO protein lysates, organoids were incubated for 1 h on ice in Cell Recovery solution (Corning, 354253) and lysed using RIPA buffer (Boston Bioproducts) supplemented with protease and phosphatase inhibitors (Roche). Protein concentration was determined by BCA assay (Thermo Fisher). Quantified protein samples were separated by electrophoresis on 4–20% ready-made Tris-Glycine gels (Invitrogen) and transferred to PVDF membranes (Millipore). Membranes were blocked with 2% bovine serum albumin for 1 h and incubated overnight with one or more primary antibodies: PSAT1 (Thermo Fisher, PA5-22124.), PHGDH (Sigma, HPA021241), PSPH (Santa Cruz, sc-365183), ERα (Cell Signaling, 8644) and Actin (Sigma, A1978). Overexpression and knockout of PSAT1 (Figure 3 ) confirmed the correct band on PSAT1 western blots. Membranes were washed with tween 20-containing tris buffered saline and incubated with fluorescence- or HRP-conjugated secondary antibodies (Bio-Rad). Rhodamine-conjugated anti-tubulin was also treated as a secondary antibody (Bio-Rad, 12004166). Images were detected using a ChemiDoc MP Imaging System (Bio-Rad).
Phgdh
Manufactured by Merck Group
PHGDH is a laboratory equipment product offered by Merck Group. It is a key enzyme involved in the serine biosynthesis pathway. PHGDH catalyzes the conversion of 3-phosphoglycerate to 3-phosphohydroxypyruvate, which is an essential step in the production of the amino acid serine.
Automatically generated - may contain errors
Lab products found in correlation
Protease and phosphatase inhibitor cocktail, by Selleck Chemicals (3 mentions)
Bca assay, by Thermo Fisher Scientific (3 mentions)
Tris glycine gel, by Thermo Fisher Scientific (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Mg132, by Selleck Chemicals (3 mentions)
Chemidoc mp imaging system, by Bio-Rad (3 mentions)
Rhodamine conjugated anti tubulin, by Bio-Rad (2 mentions)
Ripa buffer, by Boston BioProducts (2 mentions)
Cell recovery solution, by Corning (2 mentions)
Hrp conjugated secondary antibody, by Bio-Rad (2 mentions)
Protease and phosphatase inhibitor, by Roche (2 mentions)
β actin, by Thermo Fisher Scientific (2 mentions)
0.45 μm nitrocellulose membrane, by GE Healthcare (1 mentions)
Phgdh, by Cell Signaling Technology (1 mentions)
Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (1 mentions)
Hsp90, by Cell Signaling Technology (1 mentions)
Dc protein assay, by Bio-Rad (1 mentions)
Foxo4, by Cell Signaling Technology (1 mentions)
Glut1, by Cell Signaling Technology (1 mentions)
Ha 12ca5, by Roche (1 mentions)
12 protocols using phgdh
1
Protein Extraction and Western Blot Analysis
2
Western Blot Analysis of Protein Expression
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Tissue lysates were prepared by dounce homogenization in RIPA buffer (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate) containing protease inhibitors (Roche complete). Protein concentrations were determined by the DC protein assay (Bio-Rad). Lysates were mixed with 6× sample buffer containing β-ME and separated by SDS-PAGE using NuPAGE 4–12% Bis-Tris gels (Invitrogen), followed by transfer to 0.45 μm nitrocellulose membranes (GE Healthcare). The membranes were blocked in 5% non-fat milk in TBST, followed by immunoblotting with the following antibodies: PHGDH (Sigma-Aldrich, HPA021241-100), PHGDH (Cell Signaling, 13428)–liver only, GFP (Cell Signaling, 2956), HSP90 (Cell Signaling, 4874), and β-actin (Thermo Fisher, AM4302, clone AC-15).
3
Cell Culture and Transfection Protocol
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Human 293T, HCT116, SW480, and U2OS cells were cultured with Dulbecco's modified Eagle's medium media with 10% fetal bovine serum and antimicrobials. Human HBL1, DLD1, and BT483 cells were maintained in RPMI‐1640 medium supplemented with fetal bovine serum and antimicrobials. For transient transfection, cells were transfected with DNA using Lipofectamine 2000 (Invitrogen). Antibodies to the following epitopes and proteins were purchased from the indicated vendors: CSN6 (Enzo Life Sciences), HA (12CA5, Roche and Proteintech), ubiquitin (Zymed Laboratories), FOXO4 (Cell Signaling Technology and Santa Cruz Biotechnology) and COP1 (Bethyl Laboratories and Santa Cruz Biotechnology). Flag (M2 monoclonal antibody), PSAT1 and SHMT2 (Affinity Biosciences). PHGDH and Actin were purchased from Sigma. pPKB/Akt, PKB/Akt, and Glut1 were purchased from Cell Signaling Technology. GFP, MDM2, and Myc (mouse monoclonal 9E10) were purchased from Santa Cruz Biotechnology.
4
PHGDH Inhibition Assay Protocol
5
Western Blot Analysis of Cellular Proteins
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Whole cell extracts for Western blotting were prepared as described previously (16 (link)). Proteins were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto Immobilon-FL Polyvinylidene fluoride (PVDF) membranes (Millipore) according to standard procedures (Novex). Blots were probed with following antibodies: XRCC1 (Neomarkers, MS-1393-P0), DNA polymerase β (raised in-house and affinity-purified), α-tubulin (Sigma, T6199), MCM4 (abcam, ab4459-50), RP-A p32 (Bethyl, A300-244A), PCNA (Santa Cruz, sc-56), α-actin (Abcam, ab6276), PARP-1 (raised in-house and affinity-purified), p21 (Cell signaling, 12D1), PSAT1 (Novusbio, 21020002), PHGDH (Sigma, HPA021241), p53 (Santa Cruz, sc-126), PNKP (Abnova, H00011284-B01). Secondary antibodies conjugated with Alexa Fluor 680 (Molecular Probes) and IRDye® 800 (Rockland) fluorescent dyes were used. Detection and quantification was carried out using an Odyssey image analysis system (Li-Cor Biosciences).
6
Protein Extraction and Western Blot Analysis
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Cells were lysed in mammalian cell lysis buffer (50mM Tris pH 7.5, 150mM NaCl and 0.5% NP40) containing a protease and phosphatase inhibitor cocktail (Bimake.com ) and 1 μM MG132 (Selleckchem). To generate PDO protein lysates, organoids were incubated for 1 h on ice in Cell Recovery solution (Corning, 354253) and lysed using RIPA buffer (Boston Bioproducts) supplemented with protease and phosphatase inhibitors (Roche). Protein concentration was determined by BCA assay (Thermo Fisher). Quantified protein samples were separated by electrophoresis on 4–20% ready-made Tris-Glycine gels (Invitrogen) and transferred to PVDF membranes (Millipore). Membranes were blocked with 2% bovine serum albumin for 1 h and incubated overnight with one or more primary antibodies: PSAT1 (Thermo Fisher, PA5-22124.), PHGDH (Sigma, HPA021241), PSPH (Santa Cruz, sc-365183), ERα (Cell Signaling, 8644) and Actin (Sigma, A1978). Overexpression and knockout of PSAT1 (Figure 3 ) confirmed the correct band on PSAT1 western blots. Membranes were washed with tween 20-containing tris buffered saline and incubated with fluorescence- or HRP-conjugated secondary antibodies (Bio-Rad). Rhodamine-conjugated anti-tubulin was also treated as a secondary antibody (Bio-Rad, 12004166). Images were detected using a ChemiDoc MP Imaging System (Bio-Rad).
7
Metabolic Protein Expression in ME-sEV Treated CD8+ T Cells
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To assess alterations of metabolic protein expression induced by ME-sEV treatment, total proteins isolated from treated CD8+ T cells were subjected to western blot: PFKP (#ab204131, RRID:AB_2828009, Abcam), PHGDH (#HPA021241, RRID:AB_1855299, Sigma-Aldrich), PCK2 (#6924S, RRID:AB_10836185, Cell Signaling Technology), MCT4 (#sc-50329, RRID:AB_2189333, Santa Cruz) and GLUT1 (#PA1-46152, RRID:AB_2302087, Thermo Fisher).
8
Western Blot Analysis of Cellular Proteins
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Cells were washed and scraped in PBS, centrifuged and the pellet was lysed using RIPA buffer containing protease and phosphatase inhibitor cocktail (Thermo Scientific, Cat. # 78440). 25–30μg of protein was used for SDS PAGE. Tumor tissues were dissociated in RIPA buffer with protease and phosphatase inhibitor cocktail using a pellet pestle followed by sonication for 10 minutes. All primary antibodies were diluted in 5% milk in Tris-buffered Saline/0.1% Tween20 and incubated overnight at 4°C. The following primary antibodies were used: PHGDH (Sigma, Cat. #HPA021241), ATF4 (Cell Signaling Technologies, Cat. #11815S), pERK (T202/204) (Cell Signaling Technologies, Cat. # 9101S), ERK (Cell Signaling Technologies, Cat. # 4695S), pS6 Ribosomal protein (S235/236) (Cell Signaling Technologies, Cat. # 4858S), S6 Ribosomal protein (Cell Signaling Technologies, Cat. # 2217S), p4EBP1 (S65) (Cell Signaling Technologies, Cat. # 9451S), 4EBP1 (Cell Signaling Technologies, Cat. # 9452S), HA-Tag (Cell Signaling Technologies, Cat. # 3724S), pEIF2α (S51) (Cell Signaling Technologies, Cat. # 3398), EIF2a (Cell Signaling Technologies, Cat. # 5324), GFP (Cell Signaling Technologies, Cat. # 2956S), BRAF (Sigma, Cat. # HPA001328) and β-actin (Invitrogen, Cat. # AM4302)
9
Western Blot Analyses of Metabolic Enzymes
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Western blotting experiments were carried out as reported elsewhere [29 (link)]; primary antibodies against the following proteins were used: PHGDH (dilution 1:2000, #HPA021241, Sigma), PSAT (dilution 1:1000, #H00029968-A01, Novus Biologicals) pS6RP (dilution 1:2000, #2211, Cell Signaling), S6RP (1:2000, #2217, Cell Signaling), pEIF2 (dilution 1:1000, #9721, Cell Signaling), ATF4 (dilution 1:1000, #11815, Cell signaling) and β-Actin (dilution 1:10000, #A5441, Sigma).
10
Antibody Validation for Western Blot and IHC
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PHGDH (HPA021241, 1:2000 for western blot, 1:4000 or 1:8000 for IHC for the Norwegian and Dutch cohort, respectively) and PSPH (HPA020376, 1:1000 for western blot) antibodies were purchased from Sigma-Aldrich. The PSAT1 (CPTC-PSAT1-2, 1:500 for western blot) and PARP1 (AFFN-PARP1-17B10, 1:150 for western blot) antibodies, developed by the National Cancer Institute and EMBL MACF, respectively, were obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology, Iowa City, IA 52242. The phospho-Histone H2A.X (Ser139) (20E3, 1: 600 for IF), ATF4 (D4B8, 1:1000 for WESTERN BLOT) and β-actin (13E5, 1:5000, for western blot) antibodies were purchased from Cell Signaling Technology. Anti-p53 Antibody [DO-1]-Chip graded was obtained from Abcam (ab1101, 1:200 for IHC) while PAX8 Polyclonal antibody was obtained from Proteintech (10336-1-AP, 1:1200 for IHC). Secondary peroxidase conjugated goat anti-rabbit (111-035-003, 1:5000 for western blot (1:10 000 for β-actin western blot)) and goat anti-mouse antibodies (115-035-044, 1:10,000 for western blot) were purchased from Jackson ImmunoResearch. Secondary donkey anti-rabbit Alexa Fluor 594 (1:800) was purchased from Molecular Probes, Life Technologies.
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