Journal: Cells

Article Title: Vitamin D Receptor Expression Limits the Angiogenic and Inflammatory Properties of Retinal Endothelial Cells

doi: 10.3390/cells12020335

Figure Lengend Snippet: Isolation and characterization of mouse retinal EC. Vdr +/+ and Vdr −/− retinal EC were prepared and cultured as described in Materials and Methods. ( A ) Morphology of cells at subconfluent and confluent. ( B ) Vitamin D receptor expression levels were accessed by qPCR analysis (n = 3, *** p < 0.001). ( C ) Cells were incubated with medium alone (cells), 0.41% ethanol (vehicle), or 10 µM calcitriol for 24 h and VDR levels were measured by Western blot analysis. ( D ) Expression levels of EC markers were determined by flow cytometry. VE-cad; VE-cadherin and PECAM-1; Platelet endothelial cell adhesion molecule-1. Relative median fluorescent intensities are indicated in the top left corner of each panel. These cells were also positive for B4-lectin (a mouse EC specific lectin) and lacked the expression of PDGFRβ and smooth muscle actin (pericyte markers; not shown).

Article Snippet: The primary antibodies used were rat anti-PECAM-1 (553370; BD Bioscience) and rat anti-VE-cadherin (550548; BD Bioscience).

Techniques: Isolation, Cell Culture, Expressing, Incubation, Western Blot, Flow Cytometry

Journal: Cells

Article Title: Vitamin D Receptor Expression Limits the Angiogenic and Inflammatory Properties of Retinal Endothelial Cells

doi: 10.3390/cells12020335

Figure Lengend Snippet: Cellular localization and expression levels of ZO-1, N-cadherin, VE-cadherin, p120-catenin, and β-catenin. ( A ) Retinal EC were cultured on fibronectin-coated chamber slides to confluence and stained with specific antibodies as described in . ( B , C ) Western blot analysis of junctional proteins (n = 3–4, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Article Snippet: The primary antibodies used were rat anti-PECAM-1 (553370; BD Bioscience) and rat anti-VE-cadherin (550548; BD Bioscience).

Techniques: Expressing, Cell Culture, Staining, Western Blot

Journal: Scientific Reports

Article Title: Shear stress induces endothelial-to-mesenchymal transition via the transcription factor Snail

doi: 10.1038/s41598-017-03532-z

Figure Lengend Snippet: Low shear stress induced mesenchymal genes via Snail. ( a ) HUVEC were exposed to low oscillatory (+/−4 dyn/cm 2 ) or high (13 dyn/cm 2 ) wall shear stress (WSS) using a parallel plate system. ( b ) PAEC were exposed to orbital flow to generate low (5 dyn/cm 2 ) or high (15 dyn/cm 2 ) wall shear stress. ( a , b ) After 72 h, levels of EndMT marker transcripts and VE-cadherin transcripts were quantified by qRT-PCR. The expression level at the low WSS site is presented relative to the expression at the high WSS site (normalised to 1; dotted line). Data were pooled from six independent experiments using cells from different donors and mean levels +/− SEM are shown. ( c–e ) HUVEC were exposed to orbital flow to generate low (5 dyn/cm 2 ) or high (15 dyn/cm 2 ) WSS for 72 h. ( c ) Expression of N-cadherin (green) and VE-cadherin (red) was determined by immunofluorescent staining and co-staining using DAPI (blue). Scale bar, 50 μm. The proportion of cells that expressed N-cadherin or VE-cadherin was measured. ( d ) The expression levels of N-cadherin (left) and VE-cadherin (right) were assessed by Western blotting using specific antibodies and anti-PDHX antibodies were used to control for total protein levels. Representative blots are shown. Bands were quantified by densitometry. ( e ) Expression of Snail (green) was determined by immunofluorescent staining and co-staining using DAPI (blue). Scale bar, 50 μm. Fluorescence intensity was quantified in multiple cells. ( f ) HUVEC were transfected with siRNA targeting Snail or with scrambled sequences and exposed to orbital flow for 72 h. Cells exposed to low WSS (5 dyn/cm 2 ) were collected and transcript levels of Slug, N cadherin and α-SMA were quantified by qRT-PCR. ( c – f ) Data were pooled from three independent experiments using cells from different donors and mean levels +/− SEM are shown.

Article Snippet: Antibodies targeting human and murine Snail (ab180714, Abcam), human N-cadherin (ab12221, Abcam), human VE-cadherin (555661, BDPharmingen), murine CD31 (102514, Biolegend) and Ki67 (ab15580, Abcam) were obtained commercially.

Techniques: Marker, Quantitative RT-PCR, Expressing, Staining, Western Blot, Fluorescence, Transfection

Journal: Scientific Reports

Article Title: Shear stress induces endothelial-to-mesenchymal transition via the transcription factor Snail

doi: 10.1038/s41598-017-03532-z

Figure Lengend Snippet: Snail was preferentially expressed at low shear atherosusceptible sites. ( a ) EC were freshly-isolated from low wall shear stress (WSS; inner curvature) and high WSS (outer curvature) regions of the aorta in six pigs. Levels of Snail, Slug, N-cadherin, α-SMA and VE-cadherin mRNA were quantified by qRT-PCR. The expression level at the low WSS site is presented relative to the expression at the high WSS site (normalised to 1; dotted line). Mean levels +/− SEM are shown. ( b , c ) EC at low WSS (susceptible) or high WSS (protected) regions of the aorta were studied by en face staining. ( b ) C57BL/6 mice (n = 5) were stained using anti-Snail antibodies (red), co-stained using anti-CD31 antibodies (green) and counterstained using TO-PRO-3 (DNA; blue). ( c ) TWIST1 cKO or TWIST1 fl/fl mice (n = 4 each group) were stained using anti-Snail antibodies (green) and counterstained using TO-PRO-3 (DNA; blue). Representative images (scale bar, 10 μm) and quantitation of Snail fluorescence levels (mean +/− SEM) are shown. Differences between means were assessed using a paired t-test ( b ) or two-way ANOVA ( c ).

Article Snippet: Antibodies targeting human and murine Snail (ab180714, Abcam), human N-cadherin (ab12221, Abcam), human VE-cadherin (555661, BDPharmingen), murine CD31 (102514, Biolegend) and Ki67 (ab15580, Abcam) were obtained commercially.

Techniques: Isolation, Quantitative RT-PCR, Expressing, Staining, Quantitation Assay, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: Transcriptional Co-activator LEDGF Interacts with Cdc7-Activator of S-phase Kinase (ASK) and Stimulates Its Enzymatic Activity *

doi: 10.1074/jbc.M109.036491

Figure Lengend Snippet: Identification of the LEDGF-Cdc7-ASK interaction. A, schematics showing the domain organization of LEDGF and the cTAP-tagged LEDGF-(326–530) construct. Locations of the PWWP domain, NLS, AT-hooks, and IBD of LEDGF, calmodulin binding peptide (CBP), tobacco etch virus (TEV) protease site, and the IgG binding module from S. aureus protein A (protA) of the cTAP tag are indicated. B, co-IP experiments. HeLa cells were transiently transfected with HA-tagged mouse LEDGF (mLEDGF), human LEDGF, LEDGF-(326–530), HRP2, mouse p52 (mp52), or an empty vector. Whole cell extracts (WCE; lanes 1–6) or proteins pulled down with anti-HA affinity matrix from whole cell extracts (lanes 7–12) were tested by Western blotting using anti-HA, anti-Cdc7, and anti-β-actin antibodies. Migration positions of protein molecular mass standards (kDa), and the heavy chain of mouse IgG (IgG H) are indicated. C, IP of endogenous proteins. Extracts from untransfected 293T cells were incubated with rabbit anti-LEDGF antibody (lane 3) or control rabbit IgG (lane 4) and protein G-agarose, and the recovered proteins were analyzed by Western blotting with anti-Cdc7 and anti-ASK antibodies. Lanes 1 and 2 contained whole cell extract. To improve detection of ASK, the samples in lanes 2–4 were treated with λ-protein phosphatase (λPPase). The bands corresponding to ASK are indicated with asterisks.

Article Snippet: Antibodies The following primary antibodies were used in this work: mouse anti-HA (HA.11, Covance), mouse anti-Cdc7 (Lab Vision Corp.), mouse anti-β-actin (Sigma), mouse monoclonal anti-ASK (H00010926-M01, Abnova), mouse anti-LEDGF (Western blotting, clone 26, BD Biosciences), rabbit anti-LEDGF/p75 (immunoprecipitation (IP) and Western blotting, A300–848A, Bethyl Laboratories), rabbit control IgG (IP, ab46540, Abcam), mouse anti-His 5 (Qiagen), mouse anti-FLAG (M2, Sigma), rabbit anti-phospho-Ser 53 MCM2 (A300-756A, Bethyl Laboratories), rabbit anti-β tubulin (H235, Santa Cruz Biotechnology, Inc. (Santa Cruz, CA)), and goat anti-lamin B (M-20, Santa Cruz Biotechnology, Inc.).

Techniques: Construct, Binding Assay, Co-Immunoprecipitation Assay, Transfection, Plasmid Preparation, Western Blot, Migration, Incubation

Journal: The Journal of Biological Chemistry

Article Title: Transcriptional Co-activator LEDGF Interacts with Cdc7-Activator of S-phase Kinase (ASK) and Stimulates Its Enzymatic Activity *

doi: 10.1074/jbc.M109.036491

Figure Lengend Snippet: Intracellular distribution of overexpressed LEDGF, ASK and Cdc7. A, confocal laser-scanning microscopy images of HeLa cells transfected with EGFP-Cdc7, full-length HA-ASK (top and middle rows of images), HA-ASK-(1–624) (bottom row), HcRed1-LEDGF (top and bottom rows), and/or HcRed1-p52 (middle row). B, ASK and Cdc7 are enriched in the chromatin-containing Triton-insoluble fraction when co-overexpressed with LEDGF. 293T cells were transfected with expression vectors for FLAG-Cdc7 and HA-ASK (lanes w1–w4, s1–s4, and i1–i4), WT LEDGF (lanes w2, s2, and i2), LEDGF K401E/K402E/R405E (EEE) (lanes w3, s3, and i3), LEDGFΔIBD (lanes w4, s4, and i4), or empty vector (lane w0). Whole cell extracts (lanes w0–w4) and Triton X-100-soluble (lanes s1–s4), and -insoluble (lanes i1–i4) fractions were analyzed by Western blotting using anti-HA, Cdc7, LEDGF, lamin B, and β-tubulin antibodies.

Article Snippet: Antibodies The following primary antibodies were used in this work: mouse anti-HA (HA.11, Covance), mouse anti-Cdc7 (Lab Vision Corp.), mouse anti-β-actin (Sigma), mouse monoclonal anti-ASK (H00010926-M01, Abnova), mouse anti-LEDGF (Western blotting, clone 26, BD Biosciences), rabbit anti-LEDGF/p75 (immunoprecipitation (IP) and Western blotting, A300–848A, Bethyl Laboratories), rabbit control IgG (IP, ab46540, Abcam), mouse anti-His 5 (Qiagen), mouse anti-FLAG (M2, Sigma), rabbit anti-phospho-Ser 53 MCM2 (A300-756A, Bethyl Laboratories), rabbit anti-β tubulin (H235, Santa Cruz Biotechnology, Inc. (Santa Cruz, CA)), and goat anti-lamin B (M-20, Santa Cruz Biotechnology, Inc.).

Techniques: Confocal Laser Scanning Microscopy, Transfection, Expressing, Plasmid Preparation, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Transcriptional Co-activator LEDGF Interacts with Cdc7-Activator of S-phase Kinase (ASK) and Stimulates Its Enzymatic Activity *

doi: 10.1074/jbc.M109.036491

Figure Lengend Snippet: The interaction with Cdc7-ASK is mediated by the IBD of LEDGF. A, schematic of the LEDGF deletion mutants used (left) and their properties in terms of binding to and phosphorylation by Cdc7-ASK in vitro (right). B, S-tag pull-down experiments. S-tagged Cdc7-ASK was incubated with S-protein-agarose and full-length LEDGF-(1–530, lanes 6 and 7), LEDGFΔIBD (lanes 8 and 9), LEDGF-(146–530) (lanes 10 and 11), or LEDGF-(347–530) (lanes 12 and 13) in the absence (lanes 6, 8, 10, and 12) or presence (lanes 7, 9, 11, and 13) of 4 mm ATP. Proteins bound to the beads were separated by SDS-PAGE and visualized by staining with Coomassie Blue (top) and Western blotting (WB) with rabbit anti-LEDGF antibody recognizing the C terminus of LEDGF. Lanes 1–5 contained input quantities of Cdc7-ASK (lane 1), full-length LEDGF (lane 2), LEDGFΔIBD (lane 3), LEDGF-(146–530) (lane 4), or LEDGF-(347–530) (lane 5). Migration positions of molecular mass markers (kDa), LEDGF, LEDGF-(146–530), LEDGF-(347–530), ASK, Cdc7, and their hyperphosphorylated forms (ASK-P and Cdc7-P) are indicated. Stars indicate band of full-length LEDGF on the Coomassie-stained gel. C, the IBD is sufficient for the interaction with Cdc7-ASK. Cdc7-ASK was incubated with glutathione-Sepharose beads preloaded with GST-LEDGF-(347–471) (lane 2), GST-HRP2-(470–593) (lane 3), or GST (lane 4). Proteins bound to the resin were tested by Western blotting with anti-Cdc7 or anti-His5 antibodies. Lane 5 contains a mock pull-down in the absence of a GST protein. Lane 1 contained input quantity of Cdc7-ASK.

Article Snippet: Antibodies The following primary antibodies were used in this work: mouse anti-HA (HA.11, Covance), mouse anti-Cdc7 (Lab Vision Corp.), mouse anti-β-actin (Sigma), mouse monoclonal anti-ASK (H00010926-M01, Abnova), mouse anti-LEDGF (Western blotting, clone 26, BD Biosciences), rabbit anti-LEDGF/p75 (immunoprecipitation (IP) and Western blotting, A300–848A, Bethyl Laboratories), rabbit control IgG (IP, ab46540, Abcam), mouse anti-His 5 (Qiagen), mouse anti-FLAG (M2, Sigma), rabbit anti-phospho-Ser 53 MCM2 (A300-756A, Bethyl Laboratories), rabbit anti-β tubulin (H235, Santa Cruz Biotechnology, Inc. (Santa Cruz, CA)), and goat anti-lamin B (M-20, Santa Cruz Biotechnology, Inc.).

Techniques: Binding Assay, In Vitro, Incubation, SDS Page, Staining, Western Blot, Migration

Journal: The Journal of Biological Chemistry

Article Title: Transcriptional Co-activator LEDGF Interacts with Cdc7-Activator of S-phase Kinase (ASK) and Stimulates Its Enzymatic Activity *

doi: 10.1074/jbc.M109.036491

Figure Lengend Snippet: Residues 625–674 of ASK are required for binding to LEDGF. A, schematic of ASK truncations. Locations of the N, M, and C motifs are indicated. B, C terminus of ASK is required for the interaction with LEDGF. S-tagged Cdc7-ASK or its indicated mutant forms were incubated with S-protein-agarose in the presence (lanes 6–9) or absence (lanes 10–13) of LEDGF. Lane 14 contains a mock pull-down of LEDGF with S-protein-agarose. Input quantities of proteins (lanes 1–5) or proteins captured on the beads (lanes 6–14) separated by SDS-PAGE were stained with Coomassie Blue (top) and analyzed by Western blotting using anti-LEDGF antibody (bottom). C, deletion of 50 residues from the C terminus of ASK is sufficient to ablate the interaction with LEDGF. Cdc7-ASK or its mutants were incubated with S-protein-agarose beads in the presence (lanes 6–9) or absence (lanes 10–13) of LEDGF. LEDGF was incubated with S-protein-agarose alone in lane 14. Lanes 1–5 contained input levels of the indicated proteins. Samples were analyzed as in B.

Article Snippet: Antibodies The following primary antibodies were used in this work: mouse anti-HA (HA.11, Covance), mouse anti-Cdc7 (Lab Vision Corp.), mouse anti-β-actin (Sigma), mouse monoclonal anti-ASK (H00010926-M01, Abnova), mouse anti-LEDGF (Western blotting, clone 26, BD Biosciences), rabbit anti-LEDGF/p75 (immunoprecipitation (IP) and Western blotting, A300–848A, Bethyl Laboratories), rabbit control IgG (IP, ab46540, Abcam), mouse anti-His 5 (Qiagen), mouse anti-FLAG (M2, Sigma), rabbit anti-phospho-Ser 53 MCM2 (A300-756A, Bethyl Laboratories), rabbit anti-β tubulin (H235, Santa Cruz Biotechnology, Inc. (Santa Cruz, CA)), and goat anti-lamin B (M-20, Santa Cruz Biotechnology, Inc.).

Techniques: Binding Assay, Mutagenesis, Incubation, SDS Page, Staining, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Transcriptional Co-activator LEDGF Interacts with Cdc7-Activator of S-phase Kinase (ASK) and Stimulates Its Enzymatic Activity *

doi: 10.1074/jbc.M109.036491

Figure Lengend Snippet: The LEDGF-Cdc7-ASK interaction requires autophosphorylation of the kinase and the positively charged patch on the surface of the IBD structure. A, the phosphorylation state of Cdc7-ASK affects its interaction with LEDGF. Cdc7-ASK, either untreated (lanes 3 and 4), preincubated with 4 mm ATP (lanes 5 and 6), or dephosphorylated with λ-protein phosphatase (lanes 7 and 8), was incubated with protein S-agarose in the presence (lanes 4, 6, and 8) or absence (lanes 3, 5, and 7) of LEDGF. Proteins captured on the beads were separated by SDS-PAGE and detected by staining with Coomassie Blue (top) and Western blotting (WB) with anti-LEDGF antibody (bottom). B, the positive patch on the surface of the IBD is important for the interaction with Cdc7-ASK. S-tagged Cdc7-ASK was incubated with S-protein agarose in the presence of WT or mutant LEDGF. Lanes 1–5, 12, and 13 show input levels of indicated proteins. LEDGF and Cdc7-ASK were omitted from the samples in lanes 17 and 18, respectively. The samples were analyzed as in A.

Article Snippet: Antibodies The following primary antibodies were used in this work: mouse anti-HA (HA.11, Covance), mouse anti-Cdc7 (Lab Vision Corp.), mouse anti-β-actin (Sigma), mouse monoclonal anti-ASK (H00010926-M01, Abnova), mouse anti-LEDGF (Western blotting, clone 26, BD Biosciences), rabbit anti-LEDGF/p75 (immunoprecipitation (IP) and Western blotting, A300–848A, Bethyl Laboratories), rabbit control IgG (IP, ab46540, Abcam), mouse anti-His 5 (Qiagen), mouse anti-FLAG (M2, Sigma), rabbit anti-phospho-Ser 53 MCM2 (A300-756A, Bethyl Laboratories), rabbit anti-β tubulin (H235, Santa Cruz Biotechnology, Inc. (Santa Cruz, CA)), and goat anti-lamin B (M-20, Santa Cruz Biotechnology, Inc.).

Techniques: Incubation, SDS Page, Staining, Western Blot, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: Transcriptional Co-activator LEDGF Interacts with Cdc7-Activator of S-phase Kinase (ASK) and Stimulates Its Enzymatic Activity *

doi: 10.1074/jbc.M109.036491

Figure Lengend Snippet: LEDGF is phosphorylated by Cdc7-ASK in vitro. A, full-length LEDGF (lane 1), its N-terminal truncation mutants (lanes 2–6), LEDGFΔIBD (lane 7), GST-MCM2-(1–287) (lane 9), or GST (lane 10) was incubated with Cdc7-ASK in the presence of [γ-32P]ATP. Reaction products, separated by SDS-PAGE, were detected by phosphorescence imaging. Full-length LEDGF was incubated with [γ-32P]ATP in the absence of Cdc7-ASK in lane 8 (mock); Cdc7-ASK was incubated with [γ-32P]ATP in the absence of protein substrates in lane 11. Concentrations of full-length LEDGF, LEDGF deletion mutants, and GST were adjusted to 0.3 μm; GST-MCM2-(1–287) was used at 0.4 μm. Quantification of radioactivity incorporation, relative to the full-length LEDGF, is shown to the right of the gel. B, LEDGF residue Ser-206 is the major Cdc7-ASK phosphorylation target in vitro. WT LEDGF, S206A, S208A, AEA, ADA, AEA/ADA, or EEE LEDGF mutants were incubated with Cdc7-ASK in the presence of [γ-32P]ATP. The gel was stained with Coomassie Blue, and the reaction products were detected and quantified as in A.

Article Snippet: Antibodies The following primary antibodies were used in this work: mouse anti-HA (HA.11, Covance), mouse anti-Cdc7 (Lab Vision Corp.), mouse anti-β-actin (Sigma), mouse monoclonal anti-ASK (H00010926-M01, Abnova), mouse anti-LEDGF (Western blotting, clone 26, BD Biosciences), rabbit anti-LEDGF/p75 (immunoprecipitation (IP) and Western blotting, A300–848A, Bethyl Laboratories), rabbit control IgG (IP, ab46540, Abcam), mouse anti-His 5 (Qiagen), mouse anti-FLAG (M2, Sigma), rabbit anti-phospho-Ser 53 MCM2 (A300-756A, Bethyl Laboratories), rabbit anti-β tubulin (H235, Santa Cruz Biotechnology, Inc. (Santa Cruz, CA)), and goat anti-lamin B (M-20, Santa Cruz Biotechnology, Inc.).

Techniques: In Vitro, Incubation, SDS Page, Imaging, Radioactivity, Staining

Journal: The Journal of Biological Chemistry

Article Title: Transcriptional Co-activator LEDGF Interacts with Cdc7-Activator of S-phase Kinase (ASK) and Stimulates Its Enzymatic Activity *

doi: 10.1074/jbc.M109.036491

Figure Lengend Snippet: LEDGF stimulates Cdc7-ASK kinase activity in vitro. A, WT Cdc7-ASK or its mutant forms (3 nm) were incubated with 0.4 μm GST-MCM2-(1–287) and [γ-32P]ATP in the absence (lanes 1–4) or presence (lanes 5–8) of 0.4 μm LEDGF. Reaction products, separated by SDS-PAGE, were detected by phosphorescence imaging. B, WT Cdc7-ASK (lanes 1–9) or Cdc7-ASK(174–350) (lanes 10–14) was incubated with GST-MCM2-(1–287) and [γ-32P]ATP in the absence (lanes 1 and 10) or presence of the indicated concentrations of WT (lanes 2–5 and 11–14) or EEE (lanes 6–9) LEDGF.

Article Snippet: Antibodies The following primary antibodies were used in this work: mouse anti-HA (HA.11, Covance), mouse anti-Cdc7 (Lab Vision Corp.), mouse anti-β-actin (Sigma), mouse monoclonal anti-ASK (H00010926-M01, Abnova), mouse anti-LEDGF (Western blotting, clone 26, BD Biosciences), rabbit anti-LEDGF/p75 (immunoprecipitation (IP) and Western blotting, A300–848A, Bethyl Laboratories), rabbit control IgG (IP, ab46540, Abcam), mouse anti-His 5 (Qiagen), mouse anti-FLAG (M2, Sigma), rabbit anti-phospho-Ser 53 MCM2 (A300-756A, Bethyl Laboratories), rabbit anti-β tubulin (H235, Santa Cruz Biotechnology, Inc. (Santa Cruz, CA)), and goat anti-lamin B (M-20, Santa Cruz Biotechnology, Inc.).

Techniques: Activity Assay, In Vitro, Mutagenesis, Incubation, SDS Page, Imaging

Journal: The Journal of Biological Chemistry

Article Title: Transcriptional Co-activator LEDGF Interacts with Cdc7-Activator of S-phase Kinase (ASK) and Stimulates Its Enzymatic Activity *

doi: 10.1074/jbc.M109.036491

Figure Lengend Snippet: LEDGF enhances Cdc7-ASK-dependent phosphorylation of MCM2 residue Ser-53. A, GST-MCM2-(1–287) was incubated in the absence (lane 1) or presence (lanes 2–12) of 3 nm WT or the indicated mutant forms of Cdc7-ASK; 0.4 μm LEDGF, GST-LEDGF-(347–471), GST-HRP2-(470–593), or GST was added as indicated; ATP (4 mm) was present in all reactions. Reaction products, resolved by SDS-PAGE, were detected by Western blotting (WB) with a phosphospecific anti-phospho-Ser-53 MCM2 antibody (pSer53). B, full-length MCM2, purified from HeLa cells stably expressing FLAG-tagged MCM2 using anti-FLAG affinity agarose, compared with mock-purified material from parental HeLa cells. C, phosphorylation of full-length MCM2 by Cdc7-ASK in vitro. FLAG-MCM2 was incubated in the absence (lane 3) or presence of 3 nm Cdc7-ASK (lanes 4 and 5) or Cdc7-ASK-(174–350) (lane 6); 0.3 μm LEDGF was added to the reaction in lane 5. Lane 2 contains input quantity of untreated FLAG-MCM2, and lane 1 contains an equivalent amount of the mock-purified material. Reaction products were analyzed by Western blotting with anti-phospho-Ser-53 MCM2 and anti-FLAG antibodies.

Article Snippet: Antibodies The following primary antibodies were used in this work: mouse anti-HA (HA.11, Covance), mouse anti-Cdc7 (Lab Vision Corp.), mouse anti-β-actin (Sigma), mouse monoclonal anti-ASK (H00010926-M01, Abnova), mouse anti-LEDGF (Western blotting, clone 26, BD Biosciences), rabbit anti-LEDGF/p75 (immunoprecipitation (IP) and Western blotting, A300–848A, Bethyl Laboratories), rabbit control IgG (IP, ab46540, Abcam), mouse anti-His 5 (Qiagen), mouse anti-FLAG (M2, Sigma), rabbit anti-phospho-Ser 53 MCM2 (A300-756A, Bethyl Laboratories), rabbit anti-β tubulin (H235, Santa Cruz Biotechnology, Inc. (Santa Cruz, CA)), and goat anti-lamin B (M-20, Santa Cruz Biotechnology, Inc.).

Techniques: Incubation, Mutagenesis, SDS Page, Western Blot, Purification, Stable Transfection, Expressing, In Vitro

Journal: The Journal of Biological Chemistry

Article Title: Transcriptional Co-activator LEDGF Interacts with Cdc7-Activator of S-phase Kinase (ASK) and Stimulates Its Enzymatic Activity *

doi: 10.1074/jbc.M109.036491

Figure Lengend Snippet: The interaction between LEDGF IBD and the C terminus of ASK relieves autoinhibition of Cdc7-ASK kinase activity. Schematic of the Cdc7-ASK kinase activity in the absence (A) or presence (B) of LEDGF. Cdc7 is shown as a gray rectangle. ASK domains are shown as black circles; the smaller circle is the C-terminal regulatory peptide (RP). Ovals represent LEDGF PWWP and IBD domains; the MCM2–7 protein complex is shown as hexagons (P, phosphorylation). Interaction with LEDGF results in full activation of Cdc7-ASK kinase activity.

Article Snippet: Antibodies The following primary antibodies were used in this work: mouse anti-HA (HA.11, Covance), mouse anti-Cdc7 (Lab Vision Corp.), mouse anti-β-actin (Sigma), mouse monoclonal anti-ASK (H00010926-M01, Abnova), mouse anti-LEDGF (Western blotting, clone 26, BD Biosciences), rabbit anti-LEDGF/p75 (immunoprecipitation (IP) and Western blotting, A300–848A, Bethyl Laboratories), rabbit control IgG (IP, ab46540, Abcam), mouse anti-His 5 (Qiagen), mouse anti-FLAG (M2, Sigma), rabbit anti-phospho-Ser 53 MCM2 (A300-756A, Bethyl Laboratories), rabbit anti-β tubulin (H235, Santa Cruz Biotechnology, Inc. (Santa Cruz, CA)), and goat anti-lamin B (M-20, Santa Cruz Biotechnology, Inc.).

Techniques: Activity Assay, Activation Assay

Journal: bioRxiv

Article Title: Epithelial Vegfa specifies a distinct endothelial population in the mouse lung

doi: 10.1101/840033

Figure Lengend Snippet: (A) Comparison of cell dissociation protocols. For the first condition, 1.5 mL of 0.25% Trypsin-EDTA was added after collagenase digestion and the mixture was digested for an additional 15 min at 37 °C. The optimal condition (boxed) yields the highest number of viable cells in two independent experiments. NA, not available. (B) Sorting plots of an adult (>8 week-old) mouse lung with genetically labeled ECs. Left two panels: comparison of antibody staining with native fluorescence from tdT (red contour), which colocalizes mostly with CD31 and ICAM2, but also a subset of CD45 cells, perhaps due to inadvertent labeling of hematopoietic cells by Cdh5-CreER . Right two panels: for all sorting experiments, CD45 negative selection was first performed and followed by ICAM2 positive selection (red box), which labels the same ECs as CD31 but has better separation from the corresponding negative ECs.

Article Snippet: The following antibodies were used: rabbit anti-Aquaporin 5 (AQP5, 1:2500, ab78486, Abcam), goat anti-Carbonic anhydrase IV (CAR4, 1:500, AF2414, R&D), BV786 rat anti-CD31 (1:250, 740870, BD Biosciences), PE/Cy7 rat anti-CD45 (1:250, 103114, BioLegend), mouse anti-Claudin 5 (Cldn5, 1:500, Invitrogen, 352588), rabbit anti-collagen IV (COL4, 1:2500, LSL-LB-1403, CosmoBioUSA), goat anti-Delta like canonical Notch ligand 4 (DLL4, 1:250, AF1389, R&D), Alexa Fluor 488 rat anti-CD324 (ECAD, 1:500, 53-3249-80, eBioscience), goat anti-Endothelial cell specific molecule 1 (ESM1, 1:500, AF1999, R&D), rabbit anti-Avian erythroblastosis virus E-26 (v-ets) oncogene related (ERG, 1:5000, ab92513, Abcam), goat anti-Vegfr3/Flt4 (1:1000, R&D, AF743), chicken anti-beta Galactosidase (LacZ, 1:500, Ab9361, Abcam), chicken anti-Green fluorescent protein (GFP, 1:5000, AB13970, Abcam), Alexa Fluor 647 rat anti-Intercellular adhesion molecule 2 (ICAM2, 1:500, A15452, ThermoFisher), rat anti-Intercellular adhesion molecule 2 (ICAM2, 1:2500, 16-1021-82, eBioscience), goat anti-Intercellular adhesion molecule 2 (ICAM2, 1:500, AF774, R&D systems), eFluor 570 rat anti-Ki67 (1:500, 41-5698-82, eBioscience), rabbit anti-Ki67 (1:1000, RM9106S0, ThermoFisher), rabbit anti-Chondroitin sulfate proteoglycan 4 (CSPG4, 1:1000, AB5320, Millipore), rabbit anti-NK2 Homeobox 1 (NKX2.1, 1:1000, sc-13040, Santa Cruz), rat anti-Platelet derived growth factor receptor alpha (PDGFRA, 1:1000, 14-1401-82, eBioscience), goat anti-Platelet derived growth factor receptor beta (PDGFRB, 1:1000, AF1042, R&D systems), rat anti-Plasmalemma vesicle associated protein (PLVAP, 1:125, 553849, BD Biosciences), rabbit anti-Prospero Homeobox 1 (PROX1, 1:250, 11-002, AngioBio), rat anti-Advanced glycosylation end-product specific receptor (RAGE, 1:1000, MAB1179, R&D systems), rabbit anti-Red fluorescent protein (RFP, 1:1000, 600-401-379, Rockland), Cy3-conjugated mouse anti-alpha-Smooth muscle actin (SMA, 1:1000, C6198, Sigma), rabbit anti-SM22 (TAGLN, 1:2500, Abcam, ab14106), Alexa Fluor 647 rat anti-Vascular endothelial cadherin (VECAD/CDH5, 1:250, 562242, BD Biosciences), rabbit anti-Von Willebrand Factor (VWF, 1:2500, Abcam, ab6994).

Techniques: Labeling, Staining, Fluorescence, Selection

Journal: bioRxiv

Article Title: Epithelial Vegfa specifies a distinct endothelial population in the mouse lung

doi: 10.1101/840033

Figure Lengend Snippet: (A) Representative en face view of immunostaining images from at least 5 mice. Boxed region is magnified as a section view in the first three images of the bottom row. CAR4 staining covers, whereas PLVAP staining surrounds, alveolar islands (dash). Perinuclear CAR4 and PLVAP staining allows assignment of ERG nuclei to CAR4 (cyan arrowhead) versus PLVAP (magenta arrowhead) ECs, which is automatically identified, as shown in the lower rightmost image (grey nuclei are ambiguous). Scale: 10 um. (B) Wholemount immunostaining of lungs with sparsely-labeled ECs, representative of at least 5 mice, viewed as a stack (40 um), a slab (top 20 um), or a section (1 um). Accumulation of tdT to ERG nuclei allows cell numeration (1 through 5). Cell #1 is a Car4 EC and cells #2-5 are non-Car4 ECs. Line profile analysis shows aligned versus shifted peaks for Car4 versus non-Car4 ECs, respectively. For shifted peaks, Car4 ECs are closer to the airspace than non-Car4 ECs (e.g. cell #3). Asterisk, avascular tissue surrounded by a single net-like Car4 EC. Open arrow, CDH5 junction overlapping with a single Car4 EC. Cell perimeter is measured by connecting protrusions that are visible in a projection view. Tam, 0.25 ug tamoxifen. Scale: 10 um. (C) Quantification of cell perimeter and comparison using Student’s t-test.

Article Snippet: The following antibodies were used: rabbit anti-Aquaporin 5 (AQP5, 1:2500, ab78486, Abcam), goat anti-Carbonic anhydrase IV (CAR4, 1:500, AF2414, R&D), BV786 rat anti-CD31 (1:250, 740870, BD Biosciences), PE/Cy7 rat anti-CD45 (1:250, 103114, BioLegend), mouse anti-Claudin 5 (Cldn5, 1:500, Invitrogen, 352588), rabbit anti-collagen IV (COL4, 1:2500, LSL-LB-1403, CosmoBioUSA), goat anti-Delta like canonical Notch ligand 4 (DLL4, 1:250, AF1389, R&D), Alexa Fluor 488 rat anti-CD324 (ECAD, 1:500, 53-3249-80, eBioscience), goat anti-Endothelial cell specific molecule 1 (ESM1, 1:500, AF1999, R&D), rabbit anti-Avian erythroblastosis virus E-26 (v-ets) oncogene related (ERG, 1:5000, ab92513, Abcam), goat anti-Vegfr3/Flt4 (1:1000, R&D, AF743), chicken anti-beta Galactosidase (LacZ, 1:500, Ab9361, Abcam), chicken anti-Green fluorescent protein (GFP, 1:5000, AB13970, Abcam), Alexa Fluor 647 rat anti-Intercellular adhesion molecule 2 (ICAM2, 1:500, A15452, ThermoFisher), rat anti-Intercellular adhesion molecule 2 (ICAM2, 1:2500, 16-1021-82, eBioscience), goat anti-Intercellular adhesion molecule 2 (ICAM2, 1:500, AF774, R&D systems), eFluor 570 rat anti-Ki67 (1:500, 41-5698-82, eBioscience), rabbit anti-Ki67 (1:1000, RM9106S0, ThermoFisher), rabbit anti-Chondroitin sulfate proteoglycan 4 (CSPG4, 1:1000, AB5320, Millipore), rabbit anti-NK2 Homeobox 1 (NKX2.1, 1:1000, sc-13040, Santa Cruz), rat anti-Platelet derived growth factor receptor alpha (PDGFRA, 1:1000, 14-1401-82, eBioscience), goat anti-Platelet derived growth factor receptor beta (PDGFRB, 1:1000, AF1042, R&D systems), rat anti-Plasmalemma vesicle associated protein (PLVAP, 1:125, 553849, BD Biosciences), rabbit anti-Prospero Homeobox 1 (PROX1, 1:250, 11-002, AngioBio), rat anti-Advanced glycosylation end-product specific receptor (RAGE, 1:1000, MAB1179, R&D systems), rabbit anti-Red fluorescent protein (RFP, 1:1000, 600-401-379, Rockland), Cy3-conjugated mouse anti-alpha-Smooth muscle actin (SMA, 1:1000, C6198, Sigma), rabbit anti-SM22 (TAGLN, 1:2500, Abcam, ab14106), Alexa Fluor 647 rat anti-Vascular endothelial cadherin (VECAD/CDH5, 1:250, 562242, BD Biosciences), rabbit anti-Von Willebrand Factor (VWF, 1:2500, Abcam, ab6994).

Techniques: Immunostaining, Staining, Labeling