PHD1 Antibody (Rabbit mAb) [F19A8]

Application: Reactivity: Human

Usage Information

Dilution
WB1:1000-1:10000 IHC1:100 - 1:250 IF1:100 - 1:250 FCM1:10 - 1:100

Application
WB, IHC, IF, FCM

Reactivity
Human

Source
Rabbit Monoclonal Antibody

Storage Buffer
PBS, pH 7.2+50% Glycerol+0.05% BSA+0.01% NaN3

Storage (from the date of receipt)
-20°C (avoid freeze-thaw cycles), 2 years

Predicted MW
44 kDa

Datasheet & SDS

Biological Description

Specificity
PHD1 Antibody (Rabbit mAb) [F19A8] detects endogenous levels of total PHD1 protein.

Clone
F19A8

Synonym(s)
EIT6, EGLN2, Prolyl hydroxylase EGLN2, Egl nine homolog 2, Estrogen-induced tag 6, HPH-3, EIT-6, HIF-PH1, HIF-prolyl hydroxylase 1, HPH-1, PHD1

Background
PHD1, also designated EGLN2, belongs to the family of 2-oxoglutarate- and iron-dependent dioxygenases that function collectively, alongside PHD2 and PHD3, as the core oxygen-sensing machinery of metazoan cells, hydroxylating specific proline residues within a conserved LXXLAP recognition motif found in target proteins. PHD1 hydroxylates HIF1A and HIF2A at conserved proline residues located in each subunit's N-terminal and C-terminal oxygen-dependent degradation domains, and only one of these two prolines needs to be hydroxylated to trigger destruction of the HIF-alpha subunit, with hydroxylation at the C-terminal site occurring preferentially before modification at the N-terminal site. This proline hydroxylation reaction requires molecular oxygen as a direct cosubstrate, making PHD1 catalytically active only when sufficient oxygen is present, and the hydroxylated proline creates the recognition surface engaged by the von Hippel-Lindau protein, the substrate-recognition component of an Elongin BC-Cullin 2-Rbx1 E3 ubiquitin ligase complex that ubiquitinates HIF-alpha and targets it for proteasomal degradation, so that under normal oxygen tension HIF-alpha is continuously synthesized and destroyed while hypoxia removes PHD1's oxygen cosubstrate and allows HIF-alpha to escape degradation and accumulate. Beyond its canonical role in HIF regulation, PHD1 hydroxylates a distinct set of non-HIF substrates, including ATF4, IKBKB, and CEP192, extending its proline-hydroxylase activity into transcriptional and cell-cycle regulatory pathways outside the oxygen-sensing axis; PHD1-mediated hydroxylation of the transcription factor FOXO3a specifically destabilizes FOXO3a by blocking its interaction with the deubiquitinase USP9X, providing one defined mechanistic route through which PHD1 activity influences cell survival signaling independently of HIF. Ectopic expression of PHD1 suppresses HIF-1alpha accumulation and reduces secretion of vascular endothelial growth factor following hypoxic stimulation, and tumors formed by colon carcinoma cells engineered to express PHD1 show inhibited growth, increased necrosis, and reduced microvessel density when implanted in mice, directly linking restored PHD1 hydroxylase activity to suppression of the angiogenic HIF output that tumors otherwise depend on under hypoxic stress.

Background

PHD1, also designated EGLN2, belongs to the family of 2-oxoglutarate- and iron-dependent dioxygenases that function collectively, alongside PHD2 and PHD3, as the core oxygen-sensing machinery of metazoan cells, hydroxylating specific proline residues within a conserved LXXLAP recognition motif found in target proteins. PHD1 hydroxylates HIF1A and HIF2A at conserved proline residues located in each subunit's N-terminal and C-terminal oxygen-dependent degradation domains, and only one of these two prolines needs to be hydroxylated to trigger destruction of the HIF-alpha subunit, with hydroxylation at the C-terminal site occurring preferentially before modification at the N-terminal site. This proline hydroxylation reaction requires molecular oxygen as a direct cosubstrate, making PHD1 catalytically active only when sufficient oxygen is present, and the hydroxylated proline creates the recognition surface engaged by the von Hippel-Lindau protein, the substrate-recognition component of an Elongin BC-Cullin 2-Rbx1 E3 ubiquitin ligase complex that ubiquitinates HIF-alpha and targets it for proteasomal degradation, so that under normal oxygen tension HIF-alpha is continuously synthesized and destroyed while hypoxia removes PHD1's oxygen cosubstrate and allows HIF-alpha to escape degradation and accumulate. Beyond its canonical role in HIF regulation, PHD1 hydroxylates a distinct set of non-HIF substrates, including ATF4, IKBKB, and CEP192, extending its proline-hydroxylase activity into transcriptional and cell-cycle regulatory pathways outside the oxygen-sensing axis; PHD1-mediated hydroxylation of the transcription factor FOXO3a specifically destabilizes FOXO3a by blocking its interaction with the deubiquitinase USP9X, providing one defined mechanistic route through which PHD1 activity influences cell survival signaling independently of HIF. Ectopic expression of PHD1 suppresses HIF-1alpha accumulation and reduces secretion of vascular endothelial growth factor following hypoxic stimulation, and tumors formed by colon carcinoma cells engineered to express PHD1 show inhibited growth, increased necrosis, and reduced microvessel density when implanted in mice, directly linking restored PHD1 hydroxylase activity to suppression of the angiogenic HIF output that tumors otherwise depend on under hypoxic stress.

References
https://pubmed.ncbi.nlm.nih.gov/28622522/ https://pubmed.ncbi.nlm.nih.gov/14695194/

Reference Table for Selecting PVDF Membrane Pore Size Specifications

Protein Size (kDa)	PVDF Transfer Membrane Pore Size (μm)
< 10	0.22
10-20	0.22
20-30	0.45
30-45	0.45
45-70	0.45
80-100	0.45
100-140	0.45
> 140	0.45

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Protein Size (kDa)	Separating Gel Percentage
4-40	20%
12-45	15%
10-70	12.5%
15-100	10%
25-100	8%
60-210	5%