Journal: Scientific Reports

Article Title: Intrabody against prolyl hydroxylase 2 promotes angiogenesis by stabilizing hypoxia-inducible factor-1α

doi: 10.1038/s41598-019-47891-1

Figure Lengend Snippet: Expression of ER-INP in cells. ( A ) Expression of ER-INP in cells detected by RT-PCR (n = 3). ( B ) Protein expression of ER-INP in cells and the effect of ER-INP expression on the level of PHD2 protein in cells, as determined by western blot (n = 3). (C) Intracellular localization of ER-INP by immunofluorescence assay. Cells were co-transfected with pDsRed2-ER and pER-INP for 48 hours and stained with an anti-E-tag antibody to visualize the intrabody ER-INP and pDsRed2-ER as an ER marker, then observed by immunofluorescence microscopy. DsRed2-ER and ER-INP co-localized in the endoplasmic reticulum (n = 3).

Article Snippet: Anti-HIF-1α antibody (NB100-134) was purchased from Novus Biologicals (USA), anti-HIF-1α Pro402 antibody (07-1585) from Millipore (USA), anti-HIF-1α Pro564 antibody (D43B5) from Cell Signaling Technology (USA), rabbit anti-E-tag antibody (ab3397) from Abcam (UK), anti-β-tubulin antibody (K10322) from TransGen Biotech Co., LTD (Beijing, China), rabbit anti-PHD2 antibody (bs-3686R) from Bioss (Beijing, China), FITC-conjugated goat anti-rabbit IgG (HH012-1) from Beijing Dingguo Changsheng Biotechnology Co., Ltd. (Beijing, China), and mouse anti-E-tag antibody (EM50104) from HuaBio Inc. (Hangzhou, China).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Immunofluorescence, Transfection, Staining, Marker, Microscopy

Journal: Scientific Reports

Article Title: Intrabody against prolyl hydroxylase 2 promotes angiogenesis by stabilizing hypoxia-inducible factor-1α

doi: 10.1038/s41598-019-47891-1

Figure Lengend Snippet: ER-INP binds to PHD2, inhibits hydroxylation of HIF-1α and increases HIF-1α accumulation. ( A) Co-immunoprecipitation assay. HEK293 cells were transfected with control vector or intrabody ER-INP for 48 hours and subsequently lysed. Co-immunoprecipitation and western blot assays were performed on the cell lysis. ER-INP recognized and bound to PHD2 in HEK293 cells (n = 3). (B) Western blot analysis to measure the effect of ER-INP on HIF-1α and its hydroxylation level in transfected HEK293 and RAW264.7 cells pre-treated with MG132 (upper panel) and the protein ratio to β-actin loading control by ImageJ densitometry analysis (lower panel; n = 3). (C) Immunofluorescence assay. Expression of ER-INP increases HIF-1α accumulation in RAW264.7 cells. Cells were stained with anti-HIF-1α antibody and DAPI and then visualized and photographed under immunofluorescence microscopy (left), and mean fluorescence intensity of HIF-1α versus the mean fluorescence intensity of nuclear DAPI staining is displayed (right). Data represent the mean ± SD of 3 independent slides.

Article Snippet: Anti-HIF-1α antibody (NB100-134) was purchased from Novus Biologicals (USA), anti-HIF-1α Pro402 antibody (07-1585) from Millipore (USA), anti-HIF-1α Pro564 antibody (D43B5) from Cell Signaling Technology (USA), rabbit anti-E-tag antibody (ab3397) from Abcam (UK), anti-β-tubulin antibody (K10322) from TransGen Biotech Co., LTD (Beijing, China), rabbit anti-PHD2 antibody (bs-3686R) from Bioss (Beijing, China), FITC-conjugated goat anti-rabbit IgG (HH012-1) from Beijing Dingguo Changsheng Biotechnology Co., Ltd. (Beijing, China), and mouse anti-E-tag antibody (EM50104) from HuaBio Inc. (Hangzhou, China).

Techniques: Co-Immunoprecipitation Assay, Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Lysis, Immunofluorescence, Expressing, Staining, Microscopy, Fluorescence

Journal: Scientific Reports

Article Title: Intrabody against prolyl hydroxylase 2 promotes angiogenesis by stabilizing hypoxia-inducible factor-1α

doi: 10.1038/s41598-019-47891-1

Figure Lengend Snippet: Molecular mechanism of ER-INP anti-PHD2’s promotion of angiogenesis via HIF-1α stabilization and transactivation under normoxia. ER-INP intrabody binds to PHD2 and blocks PHD2 hydroxylase activity to prevent the hydroxylation, ubiquitination, and degradation of HIF-1α. HIF-1α accumulation forms dimers by binding with HIF-1β and transactivates target genes at the consensus hypoxia-responsive elements 5′-(A/G) GGTG-3′. HIF-1α target genes control numerous key cellular processes, such as angiogenesis and metabolism. HIF-1α accumulation can promote MMP-13 expression, which can also promote angiogenesis.

Article Snippet: Anti-HIF-1α antibody (NB100-134) was purchased from Novus Biologicals (USA), anti-HIF-1α Pro402 antibody (07-1585) from Millipore (USA), anti-HIF-1α Pro564 antibody (D43B5) from Cell Signaling Technology (USA), rabbit anti-E-tag antibody (ab3397) from Abcam (UK), anti-β-tubulin antibody (K10322) from TransGen Biotech Co., LTD (Beijing, China), rabbit anti-PHD2 antibody (bs-3686R) from Bioss (Beijing, China), FITC-conjugated goat anti-rabbit IgG (HH012-1) from Beijing Dingguo Changsheng Biotechnology Co., Ltd. (Beijing, China), and mouse anti-E-tag antibody (EM50104) from HuaBio Inc. (Hangzhou, China).

Techniques: Activity Assay, Binding Assay, Expressing

Journal: Cancer Research Communications

Article Title: A Novel PHD2/VHL-mediated Regulation of YAP1 Contributes to VEGF Expression and Angiogenesis

doi: 10.1158/2767-9764.CRC-21-0084

Figure Lengend Snippet: PHD2 mediates proline hydroxylation of YAP1. A, Treatment with 1 mmol/L DMOG for 24 hours elevated YAP1 protein levels in A549 and H1650 cells as compared with control vehicle treated cells, as seen by Western blotting. There was no notable change in YAP1 phosphorylation. B, Depletion of PHD2, but not PHD3, with two different siRNAs in A549 cells showed an increase in YAP1 protein levels as compared with control siRNA-transfected cells. C, Depletion of PHD2 , but not HIF1α, elevates the levels of total YAP1 in A549 cells. Depleting HIF1α along with PHD2 also elevated YAP1 levels under normoxic conditions. D, PLA revealed that YAP1 associates with PHD2 and this interaction decreases upon hypoxia (1% O 2 ) exposure for 24 hours in both A549 and H1650 cells. Scale bar, 10 μm, Blue—DAPI, Red—YAP1/PHD2 PLA foci. E, Single YAP1 or PHD2 antibody used as negative controls in the above PLA experiment. F, Co-IP assay in normoxic and hypoxic cells further confirmed that YAP1 directly interacts with PHD2 and the association is decreased in hypoxic cells. TAZ was also found to associate with PHD2 but no significant difference was observed in its levels between normoxic and hypoxic conditions. G, Depletion of PHD2 by two different siRNAs eliminates the prolyl hydroxylation of YAP1, as seen in an IP-Western blot experiment on A549 cells. H, A ubiquitin IP in PHD2 -depleted A549 cells showed a decrease in ubiquitination of YAP1 proteins compared with the control siRNA treated cells.

Article Snippet: The two different siRNAs for YAP1 were purchased from Santa Cruz Biotechnology (sc-38637) and Thermo Fisher Scientific (107951), respectively. siRNAs For TAZ (sc-45232), MST1 (sc-39249), MST2 (sc-39247), LATS2 (sc-37444), HIF1α (sc-35561), PHD2 (sc-45537), PHD3 (sc-45799), TEAD2 (sc-45232), and TEAD4 (sc-96187) were purchased from Santa Cruz Biotechnology. siRNAs for LATS1 (137593), HIF1α (106498), TAZ (122501), TEAD2 (107179), TEAD4 (115367), PHD2 (133478), and PHD3 (128749) were purchased from Thermo Fisher Scientific.

Techniques: Control, Western Blot, Phospho-proteomics, Transfection, Co-Immunoprecipitation Assay, Ubiquitin Proteomics

Journal: Cancer Research Communications

Article Title: A Novel PHD2/VHL-mediated Regulation of YAP1 Contributes to VEGF Expression and Angiogenesis

doi: 10.1158/2767-9764.CRC-21-0084

Figure Lengend Snippet: YAP1 associates with VHL E3 ligase. A, Co-IP Western blot experiments showed a direct association of YAP1 with VHL protein in both A549 and H1650 cells. B, A reverse IP confirmed the interaction of YAP1 with VHL. C, Depletion of VHL with shRNA in A549 and H1650 cells elevated YAP1 protein levels. There was marginal increase in the phosphorylation of YAP1 at pYAP1 S397 site. No change was observed in the expression of PHD2. PLA performed on A549 cells showed a decrease in the association of YAP1 with VHL during hypoxia ( D ) or in DMOG treatment ( E ). Corresponding increase in the interaction of YAP1 with HIF1α during hypoxia was observed. The right panels represent the single antibody controls for hypoxia ( D ) and DMOG ( E ) treatment. Scale bar, 25 μm. F, IP assays showed decreased association of YAP1 with VHL in the absence of PHD2. HIF1α Western blot analysis showed no change in the interaction of YAP1 with HIF1α upon PHD2 depletion. G, IP-Western blot analysis with a ubiquitin antibody in VHL -depleted cells showed a decrease in YAP1 ubiquitination as compared with shControl transfected cells. The HIF1a Western blot analysis was used as a positive control in this experiment. H, IP assays also showed decreased interaction of YAP1 with VHL in DMOG-treated A549 cells. There was also decrease in YAP1 association with PHD2 and 14-3-3 protein in the DMOG-treated cells.

Article Snippet: The two different siRNAs for YAP1 were purchased from Santa Cruz Biotechnology (sc-38637) and Thermo Fisher Scientific (107951), respectively. siRNAs For TAZ (sc-45232), MST1 (sc-39249), MST2 (sc-39247), LATS2 (sc-37444), HIF1α (sc-35561), PHD2 (sc-45537), PHD3 (sc-45799), TEAD2 (sc-45232), and TEAD4 (sc-96187) were purchased from Santa Cruz Biotechnology. siRNAs for LATS1 (137593), HIF1α (106498), TAZ (122501), TEAD2 (107179), TEAD4 (115367), PHD2 (133478), and PHD3 (128749) were purchased from Thermo Fisher Scientific.

Techniques: Co-Immunoprecipitation Assay, Western Blot, shRNA, Phospho-proteomics, Expressing, Ubiquitin Proteomics, Transfection, Positive Control

Journal: Circulation

Article Title: Inhibition of Prolyl Hydroxylase Domain Proteins Promotes Therapeutic Revascularization

doi: 10.1161/circulationaha.108.813303

Figure Lengend Snippet: Figure 2. Quantification of PHD1 (A), PHD2 (B), and PHD3 (C) mRNA (left) and protein levels (right) in shPHD- and shCON- treated mice after 2 days of treatment. D, Representative pho- tomicrographs of PHD immunostainings in ischemic muscle of mice treated with shCON, shPHD1, shPHD2, or shPHD3. Values are meanSEM; n8 per group. *P0.05, **P0.01 vs shCON ischemic (Isch); †P0.05 vs shCON nonischemic (N.Isch).

Article Snippet: Antibodies against HIF-1 , PHD1, PHD2, PHD3, carbonic anhydrase IX (1:1000; Novus Biological or Bethyl Laboratories, Interchim), VEGF-A (1:1000; Santa Cruz Biotechnology, Santa Cruz, Calif), and eNOS (1:1000; Becton Dickinson) were used for immunoblotting.

Techniques: