Journal: Scientific Reports

Article Title: Cyto-Feature Engineering: A Pipeline for Flow Cytometry Analysis to Uncover Immune Populations and Associations with Disease

doi: 10.1038/s41598-020-64516-0

Figure Lengend Snippet: Feature engineering identifies CD3+ phenotypes and correlates identified populations. ( a ) The heatmaps show the CD3+ phenotypes that constitute greater than 0.5% of the live leukocytes in at least one sample. Green indicates positive expression, and blue indicates negative expression of all 18 markers used for analysis in the flow cytometry experiment on the x-axis. The plots are separated by four different CD3+ lineages based on CD4 and CD8 expression (double negative immune cells, T helper cells, cytotoxic T cells, and double positive T cells). The “cell” column classifies cells as either central memory, effector, T stem-cell like memory, or unknown cells that need to be explored further. The “resident” column indicates if the population is a resident cell, as determined by expression of CD103. ( b ) The correlation across study samples between the percentage of cells in each population can be used to see the similarities and differences between different populations. Yellow indicates high positive correlation and purple is high negative correlation. Populations are grouped by cell lineages, and each number on the x-axis and y-axis identifies a separate cell population, corresponding to the population numbers in Fig. 3a.

Article Snippet: Thirty microliters of whole blood cells suspended in 100 μL eBioscience Flow Cytometry Staining Buffer (Cat# 00-4222-26) and stained with fluorochrome-conjugated mouse anti-human 0.5 μL CD45-PE-Cy7 (Clone: HI30), 5 μL CD3-PE (Clone: OKT3), 7 μL CD19-Alexa Fluor 488 (Clone: HIB19) and 5 μL CD27-APC (Clone: M-T271) monoclonal antibodies for twenty minutes at room temperature.

Techniques: Expressing, Flow Cytometry

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: Methods in molecular biology (Clifton, N.J.)

Article Title: Detecting Purinosome Metabolon Formation with Fluorescence Microscopy

doi: 10.1007/978-1-4939-7759-8_17

Figure Lengend Snippet: Colocalization of endogenous FGAMS and GART for visualization of purinosomes by immunofluorescence. Purine-depleted HeLa cells were fixed and permeabilized prior to being probed for with FGAMS rabbit polyclonal antibody and GART mouse monoclonal antibody. Fluorescently labeled secondary antibodies CF488A-conjugated donkey anti-rabbit and CF568-conjugated donkey anti-mouse were used to visualize the expression and localization of FGAMS and GART, respectively. A representative image of an individual cell was captured using a 100× oil objective on an Olympus Fluoview 1000 confocal laser scanning microscope. Sequential imaging of CF488A and CF568 showed colocalization of (a) FGAMS with (b) GART as represented by the yellow puncta present in (c) the merged image. Scale bar: 10 μm

Article Snippet: Primary antibody solution: 1:500 dilution of PFAS rabbit polyclonal antibody (Bethyl Laboratories) and 1:1000 dilution of GART mouse monoclonal antibody (Novus Biologicals) prepared in blocking buffer.

Techniques: Immunofluorescence, Labeling, Expressing, Laser-Scanning Microscopy, Imaging

Journal: Journal of Cancer

Article Title: Association of SMUG1 SNPs in Intron Region and Linkage Disequilibrium with Occurrence of Cervical Carcinoma and HPV Infection in Chinese Population

doi: 10.7150/jca.27103

Figure Lengend Snippet: Association between SMUG1 polymorphisms and the risk of CIN III and cervical carcinoma

Article Snippet: After blocking with 5% non-fat milk for 1h, PVDF membrane was incubated with primary mouse monoclonal antibodies: SMUG1 (1:2000) purchased from NOVUS Biologicals (Cat No. H00023583-M07) and GAPDH (1:5000) purchased from Proteintech(Cat No. Cat.60004-1-Ig) for 4°C overnight, then were washed with TBS containing 0.05%Tween-20 for three times, followed by a 1h incubation with an HRP-conjugated secondary antibody (1:5000).

Techniques: Control

Journal: Journal of Cancer

Article Title: Association of SMUG1 SNPs in Intron Region and Linkage Disequilibrium with Occurrence of Cervical Carcinoma and HPV Infection in Chinese Population

doi: 10.7150/jca.27103

Figure Lengend Snippet: Association between SMUG1 rs3087404 and rs2029167 polymorphisms with the risk of HR-HPV positive cervical carcinoma and CIN III

Article Snippet: After blocking with 5% non-fat milk for 1h, PVDF membrane was incubated with primary mouse monoclonal antibodies: SMUG1 (1:2000) purchased from NOVUS Biologicals (Cat No. H00023583-M07) and GAPDH (1:5000) purchased from Proteintech(Cat No. Cat.60004-1-Ig) for 4°C overnight, then were washed with TBS containing 0.05%Tween-20 for three times, followed by a 1h incubation with an HRP-conjugated secondary antibody (1:5000).

Techniques: Control

Journal: Journal of Cancer

Article Title: Association of SMUG1 SNPs in Intron Region and Linkage Disequilibrium with Occurrence of Cervical Carcinoma and HPV Infection in Chinese Population

doi: 10.7150/jca.27103

Figure Lengend Snippet: Association between SMUG1 rs3087404 polymorphisms and the risk for CIN III and cervical carcinoma stratified by the sexual, reproductive history

Article Snippet: After blocking with 5% non-fat milk for 1h, PVDF membrane was incubated with primary mouse monoclonal antibodies: SMUG1 (1:2000) purchased from NOVUS Biologicals (Cat No. H00023583-M07) and GAPDH (1:5000) purchased from Proteintech(Cat No. Cat.60004-1-Ig) for 4°C overnight, then were washed with TBS containing 0.05%Tween-20 for three times, followed by a 1h incubation with an HRP-conjugated secondary antibody (1:5000).

Techniques: Infection

Journal: Journal of Cancer

Article Title: Association of SMUG1 SNPs in Intron Region and Linkage Disequilibrium with Occurrence of Cervical Carcinoma and HPV Infection in Chinese Population

doi: 10.7150/jca.27103

Figure Lengend Snippet: Association between SMUG1 rs2029167 polymorphisms and the risk for CIN and cervical carcinoma stratified by the sexual, reproductive history

Article Snippet: After blocking with 5% non-fat milk for 1h, PVDF membrane was incubated with primary mouse monoclonal antibodies: SMUG1 (1:2000) purchased from NOVUS Biologicals (Cat No. H00023583-M07) and GAPDH (1:5000) purchased from Proteintech(Cat No. Cat.60004-1-Ig) for 4°C overnight, then were washed with TBS containing 0.05%Tween-20 for three times, followed by a 1h incubation with an HRP-conjugated secondary antibody (1:5000).

Techniques: Infection

Journal: Journal of Cancer

Article Title: Association of SMUG1 SNPs in Intron Region and Linkage Disequilibrium with Occurrence of Cervical Carcinoma and HPV Infection in Chinese Population

doi: 10.7150/jca.27103

Figure Lengend Snippet: Association between SMUG1 rs3087404 and rs2029167 polymorphisms and the risk for cervical carcinoma stratified by clinical pathological characteristics

Article Snippet: After blocking with 5% non-fat milk for 1h, PVDF membrane was incubated with primary mouse monoclonal antibodies: SMUG1 (1:2000) purchased from NOVUS Biologicals (Cat No. H00023583-M07) and GAPDH (1:5000) purchased from Proteintech(Cat No. Cat.60004-1-Ig) for 4°C overnight, then were washed with TBS containing 0.05%Tween-20 for three times, followed by a 1h incubation with an HRP-conjugated secondary antibody (1:5000).

Techniques:

Journal: Journal of Cancer

Article Title: Association of SMUG1 SNPs in Intron Region and Linkage Disequilibrium with Occurrence of Cervical Carcinoma and HPV Infection in Chinese Population

doi: 10.7150/jca.27103

Figure Lengend Snippet: SMUG1 mRNA expression in CSCCs with different genotypes of rs3087404 and rs2029167 (qPCR).

Article Snippet: After blocking with 5% non-fat milk for 1h, PVDF membrane was incubated with primary mouse monoclonal antibodies: SMUG1 (1:2000) purchased from NOVUS Biologicals (Cat No. H00023583-M07) and GAPDH (1:5000) purchased from Proteintech(Cat No. Cat.60004-1-Ig) for 4°C overnight, then were washed with TBS containing 0.05%Tween-20 for three times, followed by a 1h incubation with an HRP-conjugated secondary antibody (1:5000).

Techniques: Expressing

Journal: Journal of Cancer

Article Title: Association of SMUG1 SNPs in Intron Region and Linkage Disequilibrium with Occurrence of Cervical Carcinoma and HPV Infection in Chinese Population

doi: 10.7150/jca.27103

Figure Lengend Snippet: SMUG1 protein expression in CSCCs with different genotypes of rs3087404 and rs2029167 (Western Blot) (A) AA: rs3087404 genotype is AA; AG: rs3087404 genotype is AG; GG: rs3087404 genotype is GG; (B) AA: rs2029167 genotype is AA; AG: rs2029167 genotype is AG; GG: rs2029167 genotype is GG

Article Snippet: After blocking with 5% non-fat milk for 1h, PVDF membrane was incubated with primary mouse monoclonal antibodies: SMUG1 (1:2000) purchased from NOVUS Biologicals (Cat No. H00023583-M07) and GAPDH (1:5000) purchased from Proteintech(Cat No. Cat.60004-1-Ig) for 4°C overnight, then were washed with TBS containing 0.05%Tween-20 for three times, followed by a 1h incubation with an HRP-conjugated secondary antibody (1:5000).

Techniques: Expressing, Western Blot

Journal: Oncotarget

Article Title: N -acetylcysteine negatively regulates Notch3 and its malignant signaling

doi: 10.18632/oncotarget.8806

Figure Lengend Snippet: A. Time- and dose-dependent inhibition of NAC on the intracellular domain of Notch3 (N3IC), but not Notch1 (N1IC). HeLa cells were treated with NAC (2-10 mM) for 0-24 h. B. Dose-dependent inhibition by NAC (0-10 mM, 6 h) on the protein expression of N3IC in HeLa cells. C. NAC treatment (5 mM, 0-12 h) reduces protein levels of N3IC and extracellular domain of Notch 3 (N3EC) but not full length Notch 3 precursor (N3FL) in HeLa cells. Densitometry quantifications of the protein bands were shown after normalization with their respective β-actin levels. Data are presented as means ± SE, n=3. *, p < 0.05 compared with their respective non-treated group.

Article Snippet: Mouse anti-Notch3 NECD antibody (H00004854-M01, Novus Biologicals, Littleton, CO, USA) recognizes the extracellular fragments and full length Notch3 precursor.

Techniques: Inhibition, Expressing

Journal: Oncotarget

Article Title: N -acetylcysteine negatively regulates Notch3 and its malignant signaling

doi: 10.18632/oncotarget.8806

Figure Lengend Snippet: A. NAC treatment (2-10 mM, 0-24 h) decreases Hes1 and HRT1 protein levels in HeLa cells. B. NAC treatment (0-10 mM for 6 h or 5 mM for 0-12 h) decreases Hes1 and HRT1 mRNA expression in HeLa cells. The mRNA expression of NAC-treated cells was normalized to that of non-treated cells whose value was set as 1. C. NAC treatment (0-10 mM, 12 h) inhibits Hes1 reporter activity in HeLa cells. The luciferase activity in NAC-treated cells was normalized to that of non-treated cells whose value was set as 1. D. Notch3 siRNA knockdown reduces Hes1 and HRT1 levels in HeLa cells. Protein levels were determined 2 days after siRNA transfection. siCtrl, scramble siRNA; siNotch3, Notch3 siRNA. Protein densitometry quantifications were shown after normalization with β-actin levels. Data are presented as means ± SE, n=3-4. *, p < 0.05 compared with their respective non-treated group.

Article Snippet: Mouse anti-Notch3 NECD antibody (H00004854-M01, Novus Biologicals, Littleton, CO, USA) recognizes the extracellular fragments and full length Notch3 precursor.

Techniques: Expressing, Activity Assay, Luciferase, Knockdown, Transfection

Journal: Oncotarget

Article Title: N -acetylcysteine negatively regulates Notch3 and its malignant signaling

doi: 10.18632/oncotarget.8806

Figure Lengend Snippet: A. Pre-treatment with a γ-secretase inhibitor, DAPT (20 μM, 30 min), had no effect on NAC-induced (5 mM, 0-12 h) decrease in N3IC protein expression in HeLa cells. B. NAC treatment (5 mM, 0-12 h) did not affect Notch3 mRNA expression in HeLa cells. C. Pre-treatment with NH 4 Cl (25 mM, 1 h), but not lactacystin (10 μM, 30 min), reversed NAC-induced (5 mM, 0-12 h) decrease of N3IC protein levels in HeLa cells. D. NAC treatment did not affect levels of exogenously expressed Notch3 active intracellular domain (N3ICD). HeLa cells were transfected with vectors expressing N3ICD or N3FL for 24 h, followed by treatment with NAC (5 mM, 0-12 h). E. Subcellular analysis of Notch3 protein levels following NAC treatment (5 mM, 6 h) in HeLa cells. Protein levels of N3FL, N3EC and N3IC in cytosolic, nuclear and membrane fractions were determined. Successful fractionation was evidenced by using the marker proteins GAPDH, cyclin B1, and Na + , K + -ATPase. N3FL, N3EC and N3IC denoted Notch3 full length, extracellular domain and intracellular domain, respectively. Protein densitometry quantifications were shown after normalization with β-actin (A, C, D) or their respective cellular compartment markers (E). Data are presented as means ± SE, n=3-4. *, p < 0.05 compared with their respective non-treated group.

Article Snippet: Mouse anti-Notch3 NECD antibody (H00004854-M01, Novus Biologicals, Littleton, CO, USA) recognizes the extracellular fragments and full length Notch3 precursor.

Techniques: Expressing, Transfection, Membrane, Fractionation, Marker

Journal: Oncotarget

Article Title: N -acetylcysteine negatively regulates Notch3 and its malignant signaling

doi: 10.18632/oncotarget.8806

Figure Lengend Snippet: N3ICD overexpression rescues NAC-induced inhibition of proliferation (A), migration (B), and invasion (C). A. Numbers of EV- and N3ICD-transfected cells were counted at 12-48 h after NAC treatment (0-10 mM, left panel). *, p < 0.05 compared with the EV-transfected cells within the same treatment and time point. B. Results of the wound healing assay (left panels) were expressed as the migration index (the distance migrated relative to the initial scraped gap) and that of EV-transfected cells without NAC treatment was set as 100% (middle panel). C. Cells per field on the insert membrane were imaged (left panels) and counted (middle panel). B and C: *, p < 0.05 compared with no NAC treatment; #, p < 0.05 compared with the EV-transfected cells within the same treatment. Percent rescue (A-C, right panels) after N3ICD expression was calculated by dividing the net change after NAC treatment in N3ICD-transfected cells by that in EV-transfected cells. Notch3 siRNA knockdown inhibits cell proliferation D. , migration E. , and invasion F. as assessed by the same approaches described above. Representative images for migration and invasion were shown. *, p < 0.05 compared with the siCtrl-transfected cells. All data are presented as mean ±SE, n=3. I, the initial seeded cell number. EV, empty vector; N3ICD, Notch3 active intracellular domain; siCtrl, scrambled siRNA; siNotch3, Notch3 siRNA.

Article Snippet: Mouse anti-Notch3 NECD antibody (H00004854-M01, Novus Biologicals, Littleton, CO, USA) recognizes the extracellular fragments and full length Notch3 precursor.

Techniques: Over Expression, Inhibition, Migration, Transfection, Wound Healing Assay, Membrane, Expressing, Knockdown, Plasmid Preparation

Journal: Oncotarget

Article Title: N -acetylcysteine negatively regulates Notch3 and its malignant signaling

doi: 10.18632/oncotarget.8806

Figure Lengend Snippet: A. NAC treatment (5 and 10 mM, 0-24 h) decreases N3IC protein levels in HCC1937 cells. Expression of exogenous N3ICD rescues NAC-induced inhibition of proliferation B. , migration C. , and invasion D. , and Notch3 siRNA knockdown inhibits proliferation E. , migration F. , and invasion G. in HCC1937 cells. Quantifications, sample size, statistics, and abbreviations for protein levels, proliferation, migration, and invasion assays were as described in Figure & legends.

Article Snippet: Mouse anti-Notch3 NECD antibody (H00004854-M01, Novus Biologicals, Littleton, CO, USA) recognizes the extracellular fragments and full length Notch3 precursor.

Techniques: Expressing, Inhibition, Migration, Knockdown