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Anti phgdh

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Anti-PHGDH is a laboratory product that targets the enzyme 3-phosphoglycerate dehydrogenase (PHGDH). PHGDH is a key enzyme involved in the serine biosynthesis pathway. Anti-PHGDH is used in research applications to study the role of PHGDH and its implications in various biological processes.

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Lab products found in correlation

Anti β actin, by Merck Group (3 mentions) Anti calreticulin, by Cell Signaling Technology (2 mentions) Anti pkm1, by Cell Signaling Technology (2 mentions) Anti p70 s6 kinase, by Cell Signaling Technology (2 mentions) Anti pkm, by Abcam (2 mentions) Anti pkm2, by Cell Signaling Technology (2 mentions) Anti human specific ldha, by Cell Signaling Technology (2 mentions) Anti ldhb, by Abcam (2 mentions) Anti human and rabbit ldha, by Abcam (2 mentions) Anti golgin, by Cell Signaling Technology (2 mentions) Anti cox 4, by Proteintech (2 mentions) Anti gpt2, by Santa Cruz Biotechnology (2 mentions) Anti pc, by Santa Cruz Biotechnology (2 mentions) Pi31430, by Thermo Fisher Scientific (2 mentions) Pi 31460, by Thermo Fisher Scientific (2 mentions) Enhanced chemiluminescence, by Thermo Fisher Scientific (1 mentions) Anti tbk1, by Cell Signaling Technology (1 mentions) Immobilon p pvdf 0.45 μm membrane, by Merck Group (1 mentions) Nupage bis tris protein gel, by Thermo Fisher Scientific (1 mentions) Anti actin, by Santa Cruz Biotechnology (1 mentions)

6 protocols using anti phgdh

1

Western Blot Analysis of Metabolic Enzymes

Cited 2 times
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Cell lysates were prepared and subjected to WB analysis as performed previously51 (link),52 (link). The following commercial antibodies were used: anti-β-actin (Sigma A1978), anti-PKM2 (Cell Signaling Technology 3198S), anti-PKM1 (Cell Signaling Technology 7067), anti-PKM (Abcam AB118499), anti-human specific LDHA (Cell Signaling Technology 2012S), anti-LDHB (Abcam AB75167), anti-human and rabbit LDHA (Abcam AB135396), anti-p70 S6 kinase (Cell Signaling Technology 2708T), anti-calreticulin (Cell Signaling Technology 12238T), anti-golgin (Cell Signaling Technology 13192), anti-COX IV (Proteintech 11242-1-AP), anti-GPT2 (Santa Cruz sc-398383), anti-PHGDH (Sigma HPA021241), and anti-PC (Santa Cruz sc-271493), horseradish peroxidase-conjugated secondary antibodies anti-mouse (Fisher PI31430) and anti-rabbit (Fisher PI31460). All the primary antibodies were used at a 1:500 dilution in 5% non-fat milk in TBST. Secondary antibodies were used at a 1:3000 dilution in 5% non-fat milk in TBST.
Wiese E.K., Hitosugi S., Loa S.T., Sreedhar A., Andres-Beck L.G., Kurmi K., Pang Y.P., Karnitz L.M., Gonsalves W.I, & Hitosugi T. (2021). Enzymatic Activation of Pyruvate Kinase Increases Cytosolic Oxaloacetate to Inhibit the Warburg Effect. Nature metabolism, 3(7), 954-968.
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2

Western Blot Analysis of Metabolic Enzymes

Cited 2 times
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Cell lysates were prepared and subjected to WB analysis as performed previously51 (link),52 (link). The following commercial antibodies were used: anti-β-actin (Sigma A1978), anti-PKM2 (Cell Signaling Technology 3198S), anti-PKM1 (Cell Signaling Technology 7067), anti-PKM (Abcam AB118499), anti-human specific LDHA (Cell Signaling Technology 2012S), anti-LDHB (Abcam AB75167), anti-human and rabbit LDHA (Abcam AB135396), anti-p70 S6 kinase (Cell Signaling Technology 2708T), anti-calreticulin (Cell Signaling Technology 12238T), anti-golgin (Cell Signaling Technology 13192), anti-COX IV (Proteintech 11242-1-AP), anti-GPT2 (Santa Cruz sc-398383), anti-PHGDH (Sigma HPA021241), and anti-PC (Santa Cruz sc-271493), horseradish peroxidase-conjugated secondary antibodies anti-mouse (Fisher PI31430) and anti-rabbit (Fisher PI31460). All the primary antibodies were used at a 1:500 dilution in 5% non-fat milk in TBST. Secondary antibodies were used at a 1:3000 dilution in 5% non-fat milk in TBST.
Wiese E.K., Hitosugi S., Loa S.T., Sreedhar A., Andres-Beck L.G., Kurmi K., Pang Y.P., Karnitz L.M., Gonsalves W.I, & Hitosugi T. (2021). Enzymatic Activation of Pyruvate Kinase Increases Cytosolic Oxaloacetate to Inhibit the Warburg Effect. Nature metabolism, 3(7), 954-968.
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3

Western Blot Protein Detection Protocol

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For Western blot analyses, cells were lysed in a cold lysis buffer (20 mM Tris–HCl, pH 7.4, 135 mM NaCl, 1.5 mM MgCl2, 1% Triton, 10% glycerol) containing protease (Roche, UK) and phosphatase (Thermo Fisher Scientific) inhibitor cocktails. Protein content was quantified, and equal amounts (20 μg) prepared in NuPAGE LDS sample buffer (Thermo Fisher Scientific) were separated by SDS–PAGE using 4–12% NuPAGE™ Bis–Tris Protein gels (Thermo Fisher Scientific). After that, proteins were transferred to Immobilon‐P PVDF 0.45 μm membrane (Merck). Protein was detected using primary antibodies anti‐IKKɛ (1:1,000; 14907; Sigma), anti‐TBK1 (1:1,000; 3013; Cell Signaling), anti‐PHGDH (1:4,000; HPA021241; Sigma) or anti‐actin (1:2,000; sc‐1615; Santa Cruz). Enhanced chemiluminescence (Thermo Fisher Scientific) was used for signal detection.
Wilcz‐Villega E., Carter E., Ironside A., Xu R., Mataloni I., Holdsworth J., Jones W., Moreno Béjar R., Uhlik L., Bentham R.B., Godinho S.A., Dalli J., Grose R., Szabadkai G., Jones L., Hodivala‐Dilke K, & Bianchi K. (2020). Macrophages induce malignant traits in mammary epithelium via IKKε/TBK1 kinases and the serine biosynthesis pathway. EMBO Molecular Medicine, 12(2), e10491.
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4

Quantification of Protein Levels

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Protein was extracted from cells using 1X RIPA buffer (Rockland Immunochemicals, Inc.) and centrifuged at 2000 rpm for 30 minutes at 4°C. Protein concentrations were measured using Bradford Protein Assay (Bio-Rad) and loaded onto 7.5% SDS-PAGE gels transferred to PVDF membranes. Membranes were blocked in 5% dry milk in TBST and incubated with anti-β-actin (Cell Signaling 8H10D10) 1:2000 or anti-PHGDH (Sigma-Aldrich WH0026227M1) 1:1000. Horseradish peroxidase conjugated anti-mouse (Rockland 611G4302), 1:2000 was used as secondary antibody. Chemiluminescent signals were detected with Clarity Western ECL Detection Kit (Bio-Rad) and imaged using a ChemiDoc MP System (Bio-Rad).
Wang Q., Liberti M.V., Liu P., Deng X., Liu Y., Locasale J.W, & Lai L. (2016). Rational design of selective allosteric inhibitors of PHDGH and serine synthesis with in vivo activity. Cell chemical biology, 24(1), 55-65.
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5

Asparaginase-Induced Protein Expression

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Cells were cultured in either control media or media supplemented with asparaginase at 0.1 U/mL for 24 h before washing with cold PBS and lysing in a RIPA buffer. Fifty micrograms of protein were separated on 4–20% SDS-PAGE gradient gels. Proteins were transferred to an Immobilon-P membrane (Millipore Corp, Bedford, MA, USA) for an hour. Membranes were blocked in 5% skimmed milk in PBS-T and probed with primary antibodies overnight at 4 °C (anti-PHGDH- Sigma-Aldrich, anti-β-actin-Sigma-Aldrich) followed by secondary anti rabbit (Sigma-Aldrich) or anti mouse (Sigma-Aldrich).
Eraslan Z., Papatzikas G., Cazier J.B., Khanim F.L, & Günther U.L. (2021). Targeting Asparagine and Serine Metabolism in Germinal Centre-Derived B Cells Non-Hodgkin Lymphomas (B-NHL). Cells, 10(10), 2589.
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6

Western Blot Analysis of PHGDH Protein

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Cells were lysed in PhosphoSafe™ extraction reagent (EMD Millipore), followed by sonication. Cell lysates (10 μg total protein/lane loaded) were separated by SDS-PAGE and transferred onto nitrocellulose membrane. Total protein content was visualised by PonceauS staining (0.1 % (wt/vol) Ponceau S in 5 % (vol/vol) acetic acid), quantified and used as loading control. Membranes were blocked in 5 % (wt/vol) milk in Tris buffered saline with 0.1 % Tween20 (TBS-T) for one hour at RT followed by incubation overnight at 4 °C with primary antibody (anti-PHGDH; Sigma Aldrich HPA021241 and anti-GAPDH; SantaCruz biotechnology Sc-25778) diluted 1:1000 in 5 % (wt/vol) milk in TBS-T. The membranes were then washed twice in TBS-T and incubated with HRP-conjugated secondary antibody diluted 1:2000 in 5 % (wt/vol) milk in TBS-T for one hour at RT. After several washes, proteins were detected by chemiluminescence using Amersham ECL Western blotting detection reagent (GE Healthcare) according to the manufacturer’s instructions. K562 cell lysate was used as a quality control against which all samples were compared.
Unterlass J.E., Baslé A., Blackburn T.J., Tucker J., Cano C., Noble M.E, & Curtin N.J. (2016). Validating and enabling phosphoglycerate dehydrogenase (PHGDH) as a target for fragment-based drug discovery in PHGDH-amplified breast cancer. Oncotarget, 9(17), 13139-13153.
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