Journal: Journal of Biological Chemistry

Article Title: Regulation of Cellular Levels of Sprouty2 Protein by Prolyl Hydroxylase Domain and von Hippel-Lindau Proteins

doi: 10.1074/jbc.m111.303222

Figure Lengend Snippet: FIGURE 2. Endogenous PHD3 and PHD1 regulate cellular content of Spry2. HeLa cells expressing HA-Spry2 were cultured under normoxic condi- tions and transfected with control (Cont) shRNA or two shRNAs against each of PHD3 and PHD1. The siRNA experiment was as described for shRNA except cells were transfected with 20 nM control or siRNA against PHD1. Three days later, cells were lysed and analyzed for HA-Spry2, PHDs, and ERK1/2. Repre- sentatives of three experiments each are shown.

Article Snippet: All primers were synthesized by Integrated DNA Technologies Inc. Antibodies used for Western analyses of the various proteins were from the following sources: PHD1 (Novus Biological or Oxycell), PHD3 (Novus Biological or Oxycell), PHD2 (AbCam), HIF1 (BD Transduction Laboratories), ERK1/2 (Millipore), phospho-p44/p42 ERK1/2 (T202/Y204, Cell Signaling), actin (MP Biomedicals, LLC, Ohio), -PIX (Cell Signaling), HA tag (HA-HRP from Roche Applied Science), FLAG tag (FLAG-HRP from Sigma), c-Myc (Covance, monoclonal), and pVHL (Cell Signaling, polyclonal).

Techniques: Expressing, Cell Culture, Transfection, Control, shRNA

Journal: Journal of Biological Chemistry

Article Title: Regulation of Cellular Levels of Sprouty2 Protein by Prolyl Hydroxylase Domain and von Hippel-Lindau Proteins

doi: 10.1074/jbc.m111.303222

Figure Lengend Snippet: FIGURE 3. Expression of PHDs decreases cellular content of Spry2. HA-Spry2-expressing HeLa cells were transfected with empty vector or vector constructs that express FLAG-tagged PHD1, PHD2, or PHD3 and then grown in normoxia (N) (21% O2) or hypoxia (H) (3% O2) for 48 h. Cell lysates were analyzed for HA-Spry2, FLAG-PHDs, and ERK1/2 (loading control). Bar graphs show quantification of data from three experiments. Representative Western blots are shown in the bottom panels.

Article Snippet: All primers were synthesized by Integrated DNA Technologies Inc. Antibodies used for Western analyses of the various proteins were from the following sources: PHD1 (Novus Biological or Oxycell), PHD3 (Novus Biological or Oxycell), PHD2 (AbCam), HIF1 (BD Transduction Laboratories), ERK1/2 (Millipore), phospho-p44/p42 ERK1/2 (T202/Y204, Cell Signaling), actin (MP Biomedicals, LLC, Ohio), -PIX (Cell Signaling), HA tag (HA-HRP from Roche Applied Science), FLAG tag (FLAG-HRP from Sigma), c-Myc (Covance, monoclonal), and pVHL (Cell Signaling, polyclonal).

Techniques: Expressing, Transfection, Plasmid Preparation, Construct, Control, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Regulation of Cellular Levels of Sprouty2 Protein by Prolyl Hydroxylase Domain and von Hippel-Lindau Proteins

doi: 10.1074/jbc.m111.303222

Figure Lengend Snippet: FIGURE 4. Endogenous PHD1 and expressed PHDs interact with Spry2. A, HeLa cells stably expressing HA-Spry2 were grown in normoxia (N) (21% O2) or hypoxia (H) (3% O2) for 48 h. HA-Spry2 was immunoprecipitated (IP), and co-immunoprecipitated of PHD1 was monitored; nonspecific IgG was used as control. Cell lysates (WCL) were analyzed for PHD1 and Pix (loading control). IB, immunoblot. B, HeLa cells stably expressing HA-Spry2 were transfected to express FLAG- tagged PHD1, PHD2, or PHD3 and then cultured in normoxia (N) or hypoxia (H) for 24 h. The amount of HA-Spry2 that co-immunoprecipitated with FLAG-PHDs was monitoredbyWesternblots.WesternofinputshowstheexpressionofSpry2(HA)andPHDs(FLAG)inthe13,000gsupernatantusedforIP.C,sameasB,exceptthat the larger amounts of cell lysates were used to detect the co-IP of Spry2 with PHD2. Representatives of three similar experiments are shown.

Article Snippet: All primers were synthesized by Integrated DNA Technologies Inc. Antibodies used for Western analyses of the various proteins were from the following sources: PHD1 (Novus Biological or Oxycell), PHD3 (Novus Biological or Oxycell), PHD2 (AbCam), HIF1 (BD Transduction Laboratories), ERK1/2 (Millipore), phospho-p44/p42 ERK1/2 (T202/Y204, Cell Signaling), actin (MP Biomedicals, LLC, Ohio), -PIX (Cell Signaling), HA tag (HA-HRP from Roche Applied Science), FLAG tag (FLAG-HRP from Sigma), c-Myc (Covance, monoclonal), and pVHL (Cell Signaling, polyclonal).

Techniques: Stable Transfection, Expressing, Immunoprecipitation, Control, Western Blot, Transfection, Cell Culture, Co-Immunoprecipitation Assay

Journal: Journal of Biological Chemistry

Article Title: Regulation of Cellular Levels of Sprouty2 Protein by Prolyl Hydroxylase Domain and von Hippel-Lindau Proteins

doi: 10.1074/jbc.m111.303222

Figure Lengend Snippet: FIGURE7.SilencingofPHD1increasesSpry2anditsabilitytoinhibitFGF-elicitedERK1/2phosphorylation.A,controlandHA-Spry2-expressingHeLacells growninnormoxiawereserum-starvedandtreatedwithFGF(150ng/ml)forvarioustimes.CellswerelysedinLaemmlisamplebuffer,andthephosphorylation of ERK1/2 was monitored by Western analyses. B and C, same as A, except the ability of FGF to activate ERK1/2 was monitored when PHD1 was silenced in HeLa cells not expressing Spry2 (B) or in HeLa cells expressing HA-Spry2 (C). The lower panel in C shows knockdown of PHD1 expression. Total ERK1/2 was used for loading in all panels and for normalizing phospho-ERK1/2 quantification shown in D, Representative of three experiments is shown.

Article Snippet: All primers were synthesized by Integrated DNA Technologies Inc. Antibodies used for Western analyses of the various proteins were from the following sources: PHD1 (Novus Biological or Oxycell), PHD3 (Novus Biological or Oxycell), PHD2 (AbCam), HIF1 (BD Transduction Laboratories), ERK1/2 (Millipore), phospho-p44/p42 ERK1/2 (T202/Y204, Cell Signaling), actin (MP Biomedicals, LLC, Ohio), -PIX (Cell Signaling), HA tag (HA-HRP from Roche Applied Science), FLAG tag (FLAG-HRP from Sigma), c-Myc (Covance, monoclonal), and pVHL (Cell Signaling, polyclonal).

Techniques: Western Blot, Expressing, Knockdown

Journal: The Journal of Cell Biology

Article Title: VHL promotes immune response against renal cell carcinoma via NF-κB–dependent regulation of VCAM-1

doi: 10.1083/jcb.201608024

Figure Lengend Snippet: VHL and PHDs limit VCAM-1 regulation via NF-κB in ccRCC cells. (A and B) VCAM-1 mRNA (A) and protein (B) were analyzed in 786-O– or RCC4-positive or -negative cells under different stimuli for 24 h: normoxia (Nx), hypoxia 0.5% O 2 (Hp), 1 mM DMOG, or 20 µM SM7368 (SM) alone or in combination with 20 ng/ml TNF. n = 6 experiments. A representative Western blot is shown ( n = 4). As a loading control, α-tubulin was used. (C) PHD1, PHD2, PHD3, and VCAM-1 mRNA levels in 786-O-VHL cells untreated or transfected with a scrambled siRNA (Scr) or siRNAs specific for each PHD (siPHD1, siPHD2, or siPHD3) were analyzed. Gene expression is represented as fold-change over the levels of untreated cells and controlled with β-Actin as the housekeeping gene. n = 4. VCAM-1 protein levels were detected in the same samples, and densitometry quantification of protein bands controlled with α-tubulin is represented. Protein levels are expressed as fold-change over untreated cells. n = 4. A representative Western blot is shown. Statistical analysis was done using one-way ANOVA followed by Bonferroni’s posthoc test. The values represent the mean ± SEM. *, P < 0.05; **, P < 0.01; and ***, P < 0.001 were considered significant.

Article Snippet: siRNA experiments were performed with specific pools of siRNAs directed against human VCAM-1 (sc-29519), HIF-1α (sc-35561), HIF-2α (sc-35316), vhl (sc-36816), phd1 (sc-45616), phd2 (sc-45537), phd3 (sc-45799), nik (sc-36065), ikkα (sc-29365), p52 (sc-29409), and p50 (sc-29407) or with a nontargeted pool of control scrambled siRNAs (sc-37007) from Santa Cruz Biotechnology, Inc.

Techniques: Western Blot, Control, Transfection, Gene Expression