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hif1α nb100 105 antibodies  (Novus Biologicals)
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Novus Biologicals hif1α nb100 105 antibodies
DCNL1 modulates <t>HIF1α</t> activity. (A) HEK293 cells were transfected with nontargeting scrambled siRNA (siSCR) or DCNL1-targeting siRNA (siDCNL1). An equivalent amount of protein lysate was resolved by SDS-PAGE and detected by immunoblotting using the indicated antibodies. (B) Stable cell lines in the indicated cellular backgrounds were generated through lentivirus-mediated infection of two different shRNA constructs targeting DCNL1. Cells were treated with MG132 for 4 h and lysed, and an equivalent amount of protein lysate was resolved by SDS-PAGE. The indicated proteins were detected by immunoblotting. (C) The indicated stable cell lines were transiently transfected with plasmids encoding firefly luciferase under the control of a hypoxia response element and a Renilla luciferase control. Cells were treated with MG132 for 4 h prior to a dual-luciferase assay. pGIPZ was arbitrarily set to 100 to represent normal HIF1α activity. Error bars represent standard deviations of the mean. An unpaired t test was performed to assess the statistical significance.
Hif1α Nb100 105 Antibodies, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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DCNL1 modulates HIF1α activity. (A) HEK293 cells were transfected with nontargeting scrambled siRNA (siSCR) or DCNL1-targeting siRNA (siDCNL1). An equivalent amount of protein lysate was resolved by SDS-PAGE and detected by immunoblotting using the indicated antibodies. (B) Stable cell lines in the indicated cellular backgrounds were generated through lentivirus-mediated infection of two different shRNA constructs targeting DCNL1. Cells were treated with MG132 for 4 h and lysed, and an equivalent amount of protein lysate was resolved by SDS-PAGE. The indicated proteins were detected by immunoblotting. (C) The indicated stable cell lines were transiently transfected with plasmids encoding firefly luciferase under the control of a hypoxia response element and a Renilla luciferase control. Cells were treated with MG132 for 4 h prior to a dual-luciferase assay. pGIPZ was arbitrarily set to 100 to represent normal HIF1α activity. Error bars represent standard deviations of the mean. An unpaired t test was performed to assess the statistical significance.

Journal: Molecular and Cellular Biology

Article Title: DCNL1 Functions as a Substrate Sensor and Activator of Cullin 2-RING Ligase

doi: 10.1128/MCB.01342-12

Figure Lengend Snippet: DCNL1 modulates HIF1α activity. (A) HEK293 cells were transfected with nontargeting scrambled siRNA (siSCR) or DCNL1-targeting siRNA (siDCNL1). An equivalent amount of protein lysate was resolved by SDS-PAGE and detected by immunoblotting using the indicated antibodies. (B) Stable cell lines in the indicated cellular backgrounds were generated through lentivirus-mediated infection of two different shRNA constructs targeting DCNL1. Cells were treated with MG132 for 4 h and lysed, and an equivalent amount of protein lysate was resolved by SDS-PAGE. The indicated proteins were detected by immunoblotting. (C) The indicated stable cell lines were transiently transfected with plasmids encoding firefly luciferase under the control of a hypoxia response element and a Renilla luciferase control. Cells were treated with MG132 for 4 h prior to a dual-luciferase assay. pGIPZ was arbitrarily set to 100 to represent normal HIF1α activity. Error bars represent standard deviations of the mean. An unpaired t test was performed to assess the statistical significance.

Article Snippet: FLAG (catalog number NB100-63146), DCNL3 (NBP1-55423), and HIF1α (NB100-105) antibodies were obtained from Novus Biologicals.

Techniques: Activity Assay, Transfection, SDS Page, Western Blot, Stable Transfection, Generated, Infection, shRNA, Construct, Luciferase, Control

CUL2 is not required for DCNL1 binding to VHL. (A, B, and C) HEK293 cells were transfected with the indicated plasmids. Cells were treated with MG132 for 4 h. Cells were lysed, immunoprecipitated using an anti-FLAG antibody, resolved by SDS-PAGE, and immunoblotted using the indicated antibodies. (D) Bacterially purified GST or GST-DCNL1 was incubated with bacterially purified His-VHL(1–155). His pulldown (PD) was performed using nickel agarose; proteins were then resolved by SDS-PAGE and detected by immunoblotting using antibodies directed against GST and His. (E) Rabbit reticulocyte lysate in vitro translated HA-HIF1α and HA-VHL were incubated with bacterially purified GST or GST-DCNL1. GST pulldown was performed using glutathione-Sepharose. Proteins were resolved by SDS-PAGE and immunoblotted using the indicated antibodies. (F) HA-HIF1α was in vitro translated using either rabbit reticulocyte lysate or wheat germ extract and incubated with bacterially purified GST or GST-DCNL1. GST pulldown was performed using glutathione-Sepharose, and bound proteins were resolved by SDS-PAGE and detected using the indicated antibodies. WCE, whole-cell extract; IP, immunoprecipitation.

Journal: Molecular and Cellular Biology

Article Title: DCNL1 Functions as a Substrate Sensor and Activator of Cullin 2-RING Ligase

doi: 10.1128/MCB.01342-12

Figure Lengend Snippet: CUL2 is not required for DCNL1 binding to VHL. (A, B, and C) HEK293 cells were transfected with the indicated plasmids. Cells were treated with MG132 for 4 h. Cells were lysed, immunoprecipitated using an anti-FLAG antibody, resolved by SDS-PAGE, and immunoblotted using the indicated antibodies. (D) Bacterially purified GST or GST-DCNL1 was incubated with bacterially purified His-VHL(1–155). His pulldown (PD) was performed using nickel agarose; proteins were then resolved by SDS-PAGE and detected by immunoblotting using antibodies directed against GST and His. (E) Rabbit reticulocyte lysate in vitro translated HA-HIF1α and HA-VHL were incubated with bacterially purified GST or GST-DCNL1. GST pulldown was performed using glutathione-Sepharose. Proteins were resolved by SDS-PAGE and immunoblotted using the indicated antibodies. (F) HA-HIF1α was in vitro translated using either rabbit reticulocyte lysate or wheat germ extract and incubated with bacterially purified GST or GST-DCNL1. GST pulldown was performed using glutathione-Sepharose, and bound proteins were resolved by SDS-PAGE and detected using the indicated antibodies. WCE, whole-cell extract; IP, immunoprecipitation.

Article Snippet: FLAG (catalog number NB100-63146), DCNL3 (NBP1-55423), and HIF1α (NB100-105) antibodies were obtained from Novus Biologicals.

Techniques: Binding Assay, Transfection, Immunoprecipitation, SDS Page, Purification, Incubation, Western Blot, In Vitro

DCNL1 is recruited through VHL to initiate CUL2 neddylation and HIF1α degradation. (A) Schematic diagram of the indicated proteins and their capacity for recruitment of CUL2 and DCNL1. (B) The indicated constructs were in vitro translated in rabbit reticulocyte lysate and incubated with bacterially purified GST or GST-DCNL1. A GST pulldown (PD) was performed using glutathione-Sepharose, and bound proteins were resolved by SDS-PAGE and detected using the indicated antibodies. (C) HEK293 cells were transfected with the indicated plasmids. Cells were harvested at 48 h posttransfection and immunoprecipitated using an anti-GAL4 antibody. Proteins were separated by SDS-PAGE and immunoblotted using the indicated antibodies. (D) The indicated plasmids were in vitro translated, and in vitro ubiquitylation was performed in the presence (+) or absence (−) of FLAG-ubiquitin (FLAG-Ub) and S100 extracts generated from VHL-null or VHL-reconstituted cells. The reaction mixtures were immunoprecipitated using anti-GAL4 antibody, resolved by SDS-PAGE, and immunoblotted using the indicated antibodies. WCE, whole-cell extract; IP, immunoprecipitation; IB, immunoblot.

Journal: Molecular and Cellular Biology

Article Title: DCNL1 Functions as a Substrate Sensor and Activator of Cullin 2-RING Ligase

doi: 10.1128/MCB.01342-12

Figure Lengend Snippet: DCNL1 is recruited through VHL to initiate CUL2 neddylation and HIF1α degradation. (A) Schematic diagram of the indicated proteins and their capacity for recruitment of CUL2 and DCNL1. (B) The indicated constructs were in vitro translated in rabbit reticulocyte lysate and incubated with bacterially purified GST or GST-DCNL1. A GST pulldown (PD) was performed using glutathione-Sepharose, and bound proteins were resolved by SDS-PAGE and detected using the indicated antibodies. (C) HEK293 cells were transfected with the indicated plasmids. Cells were harvested at 48 h posttransfection and immunoprecipitated using an anti-GAL4 antibody. Proteins were separated by SDS-PAGE and immunoblotted using the indicated antibodies. (D) The indicated plasmids were in vitro translated, and in vitro ubiquitylation was performed in the presence (+) or absence (−) of FLAG-ubiquitin (FLAG-Ub) and S100 extracts generated from VHL-null or VHL-reconstituted cells. The reaction mixtures were immunoprecipitated using anti-GAL4 antibody, resolved by SDS-PAGE, and immunoblotted using the indicated antibodies. WCE, whole-cell extract; IP, immunoprecipitation; IB, immunoblot.

Article Snippet: FLAG (catalog number NB100-63146), DCNL3 (NBP1-55423), and HIF1α (NB100-105) antibodies were obtained from Novus Biologicals.

Techniques: Construct, In Vitro, Incubation, Purification, SDS Page, Transfection, Immunoprecipitation, Ubiquitin Proteomics, Generated, Western Blot

HIF1α(555–575) peptide has reduced capacity to promote VHL-DCNL1 interaction. The indicated constructs were in vitro translated in rabbit reticulocyte lysate and incubated with bacterially purified GST or GST-DCNL1. GST pulldown (PD) was performed using glutathione-Sepharose, and bound proteins were resolved by SDS-PAGE and detected using the indicated antibodies.

Journal: Molecular and Cellular Biology

Article Title: DCNL1 Functions as a Substrate Sensor and Activator of Cullin 2-RING Ligase

doi: 10.1128/MCB.01342-12

Figure Lengend Snippet: HIF1α(555–575) peptide has reduced capacity to promote VHL-DCNL1 interaction. The indicated constructs were in vitro translated in rabbit reticulocyte lysate and incubated with bacterially purified GST or GST-DCNL1. GST pulldown (PD) was performed using glutathione-Sepharose, and bound proteins were resolved by SDS-PAGE and detected using the indicated antibodies.

Article Snippet: FLAG (catalog number NB100-63146), DCNL3 (NBP1-55423), and HIF1α (NB100-105) antibodies were obtained from Novus Biologicals.

Techniques: Construct, In Vitro, Incubation, Purification, SDS Page