Co-immunoprecipitation was performed as previously described [15 ]. Briefly, the nuclear fraction was isolated from PC3 cells with the CelLytic NuCLEAR Extraction Kit (Sigma-Aldrich), and 50 μg of nuclear protein extracts were then precleared by incubation with 30 μl of a 50% slurry of protein A/G PLUS-Agarose beads (Santa Cruz) in a total volume of 250 μl, reconstituted with co-precipitation buffer (0.1% Triton X-100, 100 mM NaCl, 15 mM EGTA, PMSF, and a proteinase inhibitor cocktail) at 4°C for 1 h with rotation. After centrifugation the supernatants were removed and incubated with 2 μg p300 Ab (Santa Cruz) at 4°C overnight with rotation. protein A/G PLUS-Agarose beads (20 μl) were added and incubated at room temperature for 1 h with rotation. The beads were washed with wash buffer (0.1% Triton X-100, 50 mM Tris-Cl (pH 7.4), 300 mM NaCl, 5 mM EDTA) three times. SDS-PAGE sample loading buffer was added to the beads, and then boiled. Proteins were separated by SDS-PAGE, followed by western blotting with anti-p300 (Santa Cruz), anti-CREB (Millipore), anti-HIF-1α (BD Biosciences), or anti-HIF-1β/ARNT (Cell Signaling), and the protein bands were visualized by chemiluminescence (Thermo Scientific).
Anti hif 1β arnt
Manufactured by Cell Signaling Technology
Anti-HIF-1β/ARNT is a research antibody product manufactured by Cell Signaling Technology. It binds to the HIF-1β/ARNT protein, which is a key component of the hypoxia-inducible factor (HIF) transcriptional complex.
Automatically generated - may contain errors
Lab products found in correlation
Cellytic nuclear extraction kit, by Merck Group (2 mentions)
Protein a g plus agarose bead, by Santa Cruz Biotechnology (2 mentions)
Anti creb, by Merck Group (1 mentions)
Chemiluminescence, by Thermo Fisher Scientific (1 mentions)
Anti p300, by Santa Cruz Biotechnology (1 mentions)
Anti hif 1α, by BD (1 mentions)
Anti gapdh, by Novus Biologicals (1 mentions)
Ecl reagent, by Cytiva (1 mentions)
Anti lamin a c, by Cell Signaling Technology (1 mentions)
Anti bmal1, by Cell Signaling Technology (1 mentions)
Anti hif 1α, by R&D Systems (1 mentions)
Anti clock, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
2 protocols using anti hif 1β arnt
1
Nuclear Protein Co-immunoprecipitation Assay
2
Nuclear Protein Extraction and Immunoblotting
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Cells were immediately placed on ice and washed with ice-cold PBS. All wash buffers and the final resuspension buffer included 1x protease inhibitor mixture (GE Healthcare), NaCl (150 μM), β-glycerophosphate (62.5 mM), DTT (0.1 μM), NaF (5 mM), and Na3VO4 (200 μM). When needed, CelLytic NuCLEAR extraction kit (Sigma Aldrich) was used to generate nuclear proteins. Immunoprecipitation was performed using Protein A/G PLUS-Agarose beads (SantaCruz) following standard protocol. Proteins were resolved on 8–12% SDS-polyacrylamide gels and transferred by electroblotting to PVDF membranes (Bio-Rad). The membranes were blocked with 5% nonfat dry milk in TBST (50 mM Tris pH 7.6 with 0.1% Tween 20) and incubated overnight at 4°C in 5% nonfatdry milk in TBST with antibody. Immunolabeling was detected using the ECL reagent (Amersham Biosciences). Relative expression levels were determined by quantitative densitometric analysis using one-dimensional image analysis software (GE Healthsciences). Antibodies used were anti-BMAL1 (#14020, Cell Signaling), anti-CLOCK (#5157, Cell Signaling; #3517, Abcam), anti-RORα (#NBP1-52813, Novus), anti-HIF-1α (#MAB1536, R&D Systems; #ab2185, Abcam), anti-HIF-1β/ARNT (#5537, Cell Signaling), anti-GAPDH (#NB300-221SS, Novus) and anti-Lamin A/C (#2032, Cell Signaling).
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