Anti-PHD1/prolyl hydroxylase antibody [EPR2745]

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PHD1/prolyl hydroxylase antibody [EPR2745] (AB108980)

Immunohistochemical analysis of paraffin-embedded Human ovarian adenocarcinoma tissue using ab108980

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-PHD1/prolyl hydroxylase antibody [EPR2745] (AB108980)

Immunofluorescent staining of HeLa cells using ab108980

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PHD1/prolyl hydroxylase antibody [EPR2745] (AB108980)

Immunohistochemical analysis of paraffin-embedded Human testis tissue using ab108980

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-PHD1/prolyl hydroxylase antibody [EPR2745] (AB108980)

Overlay histogram showing HeLa cells stained with ab108980 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab108980 1/100 dilution) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monclonal) (1μg/1x10⁶ cells) used under the same conditions. Acquisition of >5000 events was performed.

• WB

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Western blot - Anti-PHD1/prolyl hydroxylase antibody [EPR2745] (AB108980)

All lanes:

Western blot - Anti-PHD1/prolyl hydroxylase antibody [EPR2745] (ab108980) at 1/1000 dilution

Lane 1:

A549 cell lysate at 10 µg

Lane 2:

HeLa cell lysate at 10 µg

Lane 3:

MCF-7 cell lysate at 10 µg

Secondary

All lanes:

Standard HRP labelled goat anti-rabbit at 1/2000 dilution

Predicted band size: 44 kDa

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• WB

CiteAb

Western blot - Anti-PHD1/prolyl hydroxylase antibody [EPR2745] (AB108980)

Western Blotting using Anti-PHD1/prolyl hydroxylase antibody [EPR2745], ab108980. Publication image from Hsu, T. S. et al., 2016, Nat Commun, 27312851. Legend direct from paper.

DAPK interacts with PHD2 and increases PHD2-HIF-1α association.(a) Interaction of DAPK with PHD2. Jurkat cells were transfected with FLAG-PHD2 and DAPK-Myc. The whole-cell lysates (WCL) were immunoprecipitated with anti-Myc, and the contents of FLAG-PHD2 and DAPK-Myc in the precipitates and WCL were determined by anti-FLAG and anti-Myc. (b) Co-localization of DAPK with PHD2 in Th17 cells. WT naive T cells were differentiated into Th17 for 2 days, and cells were fixed. Fixed cells were stained with anti-PHD2, anti-DAPK and DAPI, and analysed by confocal microscopy. Scale bar, 2.5 µm. (c) Co-localization of HIF-1α with PHD2 in Th17 cells. Th17 cells were stained with anti-PHD2, anti-HIF-1α and DAPI, and analysed by confocal microscopy. Scale bar, 2.5 µm. (d) DAPK interacts with the N-terminal domain of PHD2. HEK293T cells were transfected with DAPK-Myc, and the N-terminal (aa 1–180) or C-terminal (aa 181–426) domain of FLAG-PHD2 as indicated. Cell lysates were immunoprecipitated with anti-Myc, and the contents of PHD2 and DAPK were determined. (e) DAPK increases the association of PHD2 with HIF-1α in vitro. Recombinant GST-HIF-1α protein was incubated with FLAG-PHD2 in the absence or presence of recombinant DAPK-FLAG proteins in PBS at 4 °C for 4 h. The GST-HIF-1α complex was pulled down by anti-GST, and the contents of FLAG-PHD2, GST-HIF-1α and DAPK-FLAG in the precipitates and incubation mixtures were determined. Data (a–e) are representative of two independent experiments.

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• WB

CiteAb

Western blot - Anti-PHD1/prolyl hydroxylase antibody [EPR2745] (AB108980)

Western Blotting using Anti-PHD1/prolyl hydroxylase antibody [EPR2745], ab108980. Publication image from Hsu, T. S. et al., 2016, Nat Commun, 27312851. Legend direct from paper.

DAPK promotes PHD2-dependent HIF-1α degradation.(a) Deletion of the oxygen-dependent degradation (ODD) region confers resistance of HIF-1α to DAPK-induced degradation. HEK293T cells were transfected with DAPK-FLAG and HIF-1α[δODD] and the levels of HIF-1α were determined 48 h after transfection. (b) PHD inhibitor prevents DAPK-induced HIF-1α degradation in normal T cells. Naive T cells were activated for 48 h and transduced with vector or DAPK-FLAG-IRES-GFP. GFP+ T cells were sorted 48 h later, switched to hypoxic incubation (1% O2) in the absence or presence of DFX (10 µM) for another 24 h, and the levels of DAPK-FLAG and HIF-1α were determined. (c) DAPK deficiency decreases proline hydroxylation of HIF-1α in normal T cells. Naive WT and Dapk−/− T cells were activated with anti-CD3/CD28 for 48 h in normoxic conditions, followed by treatment with MG132 (10 µM). The extents of Pro564 hydroxylation on HIF-1α were determined at the indicated time points. (d) DAPK enhances proline hydroxylation of HIF-1α. YFP control and DAPK-FLAG-expressing Jurkat cells were treated with MG132 (5 µM) in normoxic conditions. The extents of Pro564 hydroxylation on HIF-1α were determined at the indicated time points. (e) Mutation of proline hydroxylation sites confers resistance of HIF-1α to DAPK-induced degradation in T cells. Jurkat cells were transfected with DAPK-FLAG, HIF-1α-WT or HIF-1α[P402A/P564A] (HIF-1α-2PA), as indicated. Twenty-four hours after transfection, cells were incubated under hypoxic conditions (1% O2) for 6 h, then switched back to normoxia for 10 min, and the levels of HIF-1α were determined. (f) DAPK deficiency does not affect the expression of PHD1 and PHD2. WT and Dapk−/− naive CD4 T cells were allowed to differentiate into Th17 cells for 3 days. Cells were harvested and the expression of PHD1 and PHD2 was examined. (g) PHD2-knockdown prevents DAPK-induced HIF-1α degradation in T cells. Jurkat T cells were transfected with DAPK-FLAG, siRNA control (siCtrl), or siRNA for PHD2 (siPHD2). Twenty-four hours later, cells were switched to hypoxic conditions for 6 h, and the levels of HIF-1α, PHD2 and HSP90 were determined. Data are representative of three (a,d) or two (b,c,e–g) independent experiments.

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