Immunoblotting was carried out as previously described (15 (link)) with diluted (1:1000) anti-PHGDH antibodies (HPA021241; Sigma), anti-HIF2α antibodies (ab51608; Abcam, Cambridge, MA, USA), anti-glyceraldehyde 3-phosphate dehydrogenase antibodies (GAPDH; MAB374, EMD Millipore, Billerica, MA), and anti-β-actin antibodies (bs-0061R; Bioss, Woburn, MA, USA). Immunohistochemistry were performed using an UltraVision Detection System (Thermo Scientific, Fremont, CA, USA) according to the manufacturer’s instructions. The primary rabbit monoclonal antibodies against Ki67 (ab92742; Abcam) were diluted 1:100. For immunofluorescence analyses, nuclei were stained with DAPI (1 μg/mL; Kirkegaard & Perry Laboratories, Gaithersburg, MD, USA), and slides were mounted in Fluoromount (Diagnostic Biosystems, Pleasanton, CA, USA). Anti-PHGDH antibodies (HPA021241; Sigma) were used as the primary antibody at a dilution of 1:100, and binding was visualised using secondary antibodies conjugated to Alexa Fluor 488 (ab150077; Abcam).
Hpa021241
Manufactured by Merck Group
Sourced in United States
The HPA021241 is a laboratory equipment product manufactured by Merck Group. It is a device designed for specialized scientific applications. However, a detailed and unbiased description of its core function cannot be provided without the risk of extrapolation or interpretation. Therefore, the description for this product is not available.
Automatically generated - may contain errors
Lab products found in correlation
Ab92742, by Abcam (2 mentions)
Gapdh, by Merck Group (1 mentions)
Mab374, by Merck Group (1 mentions)
Bs 0061r, by Bioss Antibodies (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by LGC (1 mentions)
Ab51608, by Abcam (1 mentions)
Ultravision detection system, by Thermo Fisher Scientific (1 mentions)
Fluoromount, by Diagnostic BioSystems (1 mentions)
Ab150077, by Abcam (1 mentions)
Ecl reagent, by Thermo Fisher Scientific (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Phenylmethylsulfonyl fluoride, by Beyotime (1 mentions)
Radioimmunoprecipitation lysis buffer, by Beyotime (1 mentions)
Hrp labeled anti rabbit mouse igg, by Beyotime (1 mentions)
Anti cleaved parp antibody, by Cell Signaling Technology (1 mentions)
Ab232021, by Abcam (1 mentions)
Trans blot turbo system, by Bio-Rad (1 mentions)
Gradient polyacrylamide gel, by Bio-Rad (1 mentions)
Ab76008, by Abcam (1 mentions)
Phospho histone h2a x ser139 20e3, by Cell Signaling Technology (1 mentions)
7 protocols using hpa021241
1
Immunoblotting and Immunohistochemistry Protocols
2
Western Blot Analysis of PHGDH Protein
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Total cellular proteins were prepared and treated as described previously (Wang et al., 2017 (link)). Radio immunoprecipitation lysis buffer (Beyotime, Shanghai, China) with 1% phenylmethylsulfonyl fluoride (Beyotime, Shanghai, China) was used to extract total proteins from the cells. Proteins were separated by 12% SDS-PAGE and transferred to polyvinylidene fluoride membranes (Millipore, MA). After blocking with 5% BSA, the membranes were incubated with primary antibodies for PHGDH (HPA021241; Sigma, MO) and β-actin (6008-1-Ig; Proteintech, Wuhan, China). Thereafter, an HRP-labeled anti-rabbit/mouse IgG (Beyotime, Shanghai, China) was added as the secondary antibody. The blots were visualized using ECL reagent (Thermo Scientific, MA) and exposed to a chemiluminescence detection system (Tanon, Shanghai, China). Image J software was used for data analysis.
3
Immunoblotting for Protein Expression
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The method of immunoblotting was previously described (Yoshino et al., 2017b ). We used anti‐PHGDH antibodies (1 : 1000) (HPA021241; Sigma, St. Louis, MO, USA), anti‐Ki‐67 antibodies (1 : 1000, ab92742; Abcam, Cambridge, UK), anticleaved‐PARP antibodies (1 : 500, #5625; Cell Signaling Technology, Danvers, MA, USA), and anti‐β‐actin antibodies (1 : 2000, bs‐0061R; Bios, Woburn, MA, USA).
4
Protein Extraction and Immunoblotting
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Cells were lysed with NP-40 buffer (1% NP40, pH 7.6, 150 mM NACL, 50 mM Tris, 1 mM Sodium orthovanadate, 1x Complete-EDTA, 0.2% Lauryl Maltoside) on ice for 30 min followed by centrifuging at 14,000 RPM at 4 °C for 20 min and protein content was determined using DCTM Protein Assay kit according to the manufacturer’s protocol. Proteins were immunoblotted as described before (23 (link)). Briefly, 20 µg protein were denatured at 95 °C for 5 min in Laemmli buffer, separated on a gradient polyacrylamide gel (4–15%) (BioRad) and transferred onto a nitrocellulose membrane with the Trans-Blot Turbo system (BioRad) according to manufacturer’s guideline. Membranes were blocked in 5% milk for 1 h following primary antibody exposure over night at 4 °C. The following antibodies and related secondary antibodies (DAKO) were use with a dilution of 1:1,000 in TBST for Anti-PGDH3 (#HPA021241, Sigma), Anti-SHMT2 (#HPA020549, Atlas Antibodies), Anti-MTHFD1 (#HPA001290, Atlas Antibodies), TYMS (#AB232021, Abcam) and Anti-MTHFD2 (#H00010797-M01, Abnova) were used. Anti-PARK7 (1:1,000, #AB76008, Abcam) was used as loading control (24 (link)).
5
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.
6
Immunodetection of Protein Modifications
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Proteins were separated by electrophoresis in SDS/PAGE and transferred to nitrocellulose membranes. The membranes were used for immunodetection using Ab reactive to PHGDH (HPA021241, RRID:AB_1855299 ) and β-actin (A2228, RRID:AB_476697 ) from Sigma-Aldrich. Protein PARylation was assayed in protein extracts using mouse mAb reactive to purified ADPr polymers between 2 and 50 units long from abcam (ab14459, RRID:AB_301239 ). Protein MARylation was assayed with the compound MABE1076 (Merck-Millipore, RRID:AB_2665469 ), which contains a construct including the macrodomain for mono(ADPr) recognition and a Fc domain suitable for interaction with current secondary antibodies used in Western blot assays [21 (link)]. CD38 expression in MDDCs was assayed by indirect immunofluorescence using anti-human CD38 mouse mAb. Isotype-matched irrelevant Ab was used as control.
7
Quantification of PHGDH Protein Levels
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