Protein samples (20-40 μg) from treated cell extracts were resolved by 10% or 12% SDS-PAGE gel and were transferred to a polyvinylidene fluoride membrane (Bio-Rad, Hercules, CA). The membranes were probed for various proteins using the appropriate antibodies (primary antibody incubated overnight at 4°C, secondary antibody incubated for 1 h at room temperature) and were visualized using an electrochemiluminescence (ECL) system (Thermo Fisher Scientific, Waltham, MA). The bands were imaged and analyzed using the ChemiDoc™ XRS+ System with Image Lab™ Software (Bio-Rad, Hercules, CA). The applied primary antibodies included HIF-1α (1 : 1000 Novus Biologicals, Littleton, CO), hydroxy-HIF-1α (1 : 1000 Cell Signaling Technology, Danvers, MA), ISCU1/2 (1 : 200 Santa Cruz Biotechnology, Santa Cruz, CA), aconitase 2 (1 : 1000), and NDUFA9 (1 : 2000, Abcam, Cambridge, UK).
Hydroxy hif 1α
Manufactured by Cell Signaling Technology
Sourced in United States
Hydroxy-HIF-1α is a protein that functions as a subunit of the hypoxia-inducible factor 1 (HIF-1) transcription complex. HIF-1 is a key regulator of cellular response to low oxygen conditions (hypoxia).
Automatically generated - may contain errors
Lab products found in correlation
Odyssey scanning system, by LI COR (3 mentions)
β actin, by Santa Cruz Biotechnology (3 mentions)
Hif 1α, by Novus Biologicals (2 mentions)
Hexokinase 2, by Cell Signaling Technology (2 mentions)
Sirt3, by Cell Signaling Technology (2 mentions)
Image lab software, by Bio-Rad (1 mentions)
Polyvinylidene fluoride membrane, by Bio-Rad (1 mentions)
Ecl system, by Thermo Fisher Scientific (1 mentions)
Ndufa9, by Abcam (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
Aconitase 2, by Abcam (1 mentions)
Pepck, by Santa Cruz Biotechnology (1 mentions)
G6pase, by Santa Cruz Biotechnology (1 mentions)
P akt, by Santa Cruz Biotechnology (1 mentions)
Pfkfb3, by Cell Signaling Technology (1 mentions)
Cox 2, by Santa Cruz Biotechnology (1 mentions)
C myc, by Santa Cruz Biotechnology (1 mentions)
Hif 1α, by Cell Signaling Technology (1 mentions)
Sirt1, by Cell Signaling Technology (1 mentions)
Foxo3a, by Cell Signaling Technology (1 mentions)
5 protocols using hydroxy hif 1α
1
Western Blot Analysis of Hypoxia Proteins
2
Western Blot Analysis of Cellular Proteins
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Protein samples were isolated with lysis buffer, eluted with SDS buffer, separated on SDS-polyacrylamide gels, and electroblotted onto PVDF membranes [51 (link)]. Immunoreactive protein bands were detected using an Odyssey Scanning System (LI-COR Inc., Superior St., Lincoln, NE). The following antibodies were used for Western blotting: PGM, MDM2, β-actin (Santa Cruz Biotechnology, CA) at 1:400 dilution; SIRT3, hexokinase II, hydroxy-HIF-1α, PTEN, GLUT4 (Cell signaling Technology, Inc., MA) at 1:800 dilution; SIRT3 of nuclear protein (Abcam Ltd, HK, China) at 1:1000 dilution; TIGAR (Anspec, Inc., San Jose, CA); p53 (Ab-6) (EMD Chemicals, Gibbstown, NJ).
3
Protein Expression Analysis in Metabolic Regulation
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Proteins were isolated using lysis buffer, incubated in SDS buffer, separated on SDS-polyacrylamide gels, and electroblotted onto PVDF membranes. Immunoreactive protein bands were detected using an Odyssey Scanning System (LI-COR Inc., Superior St., Lincoln, NE, USA). The following antibodies were used for Western blotting: G6Pase, FBPase, PEPCK, COX2, c-Myc, p-AKT, p-p38, PDHK, MCT1, β-actin (Santa Cruz, CA, USA) at 1:400 dilution; MCT4, p-ERK, GLUT 4, (Bioworld Technology, Inc., MN) at 1:800 dilution; HIF-1α, Hexokinase II, Hydroxy-HIF-1α, PFKFB 3 (Cell Signaling Technology, Inc., MA) at 1:800 dilution.
4
Western Blot Analysis of Hypoxia Signaling
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Proteins were isolated using lysis buffer, incubated in SDS buffer, separated on SDS-polyacrylamide gels, and electroblotted onto PVDF membranes. Immunoreactive protein bands were detected using an Odyssey Scanning System (LI-COR Inc., Superior St., Lincoln, NE, USA). The following antibodies were used for western blotting: HIF1α, PHD1, PHD2, PHD3, SOD2, β-actin (Santa Cruz Biotechnology) at 1 : 400 dilution; SIRT3, SIRT1, HKII, Hydroxy-HIF-1α, FOXO3a (Cell Signaling Technology) at 1 : 800 dilution.
5
Immunoblot Analysis of TLR Signaling Pathway
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Protein extracts were resolved by SDS-PAGE and transferred to nylon membranes (Immobilon-P; Merck KGaA) using standard techniques. Primary antibodies used for immunoblotting were as follows: MYD88 (sc-11356; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) for the detection of the N-terminal of MYD88, MYD88 (#4283; Cell Signaling Technology, Inc., Danvers, MA, USA), phospho-IRAK4 (#11927; Cell Signaling Technology, Inc.), IRAK4 (#4363; Cell Signaling Technology, Inc.), phospho-p65 (#3033; Cell Signaling Technology, Inc.), p65 (sc-372; Santa Cruz Biotechnology Inc.), phospho-IκBα (#9246; Cell Signaling Technology, Inc.), IκBα (sc-371; Santa Cruz Biotechnology, Inc.), HIF-1α (NB100-479; Novus Biologicals, Centennial, CO, USA), HIF-2α (NB100-122; Novus Biologicals), hydroxy-HIF-1α (#3434; Cell Signaling Technology, Inc.), phosphor-JNK (#9251; Cell Signaling Technology, Inc.), JNK (#9252; Cell Signaling Technology, Inc.), phosphor-ERK (#9101; Cell Signaling Technology, Inc.), ERK (#9102; Cell Signaling Technology, Inc.), phosphor-p38 (#9211; Cell Signaling Technology, Inc.), p38 (#9212; Cell Signaling Technology, Inc.), phosphor-MAPKAP2 (#3316; Cell Signaling Technology, Inc.), and MAPKAP2 (#12155; Cell Signaling Technology, Inc.).
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