Journal: Molecular Cancer Research

Article Title: The Stress Oncoprotein LEDGF/p75 Interacts with the Methyl CpG Binding Protein MeCP2 and Influences Its Transcriptional Activity

doi: 10.1158/1541-7786.mcr-11-0314

Figure Lengend Snippet: Figure 1. Identification of candidate interacting partners of LEDGF/p75 using transcription factor protein arrays. A, Coomassie blue–stained SDS-PAGE gel showing purified His-LEDGF/p75. E. coli BL21 strain was transformed with pET28a-dfs70 encoding His-LEDGF/p75 and induced with IPTG. Lysate was passed through a nickel column to purify His- LEDGF/p75. B, immunoblot showing the specificity of the human autoantibody against LEDGF/p75 used as detection reagent in the transcription factor protein arrays. The autoantibody reacts specifically with LEDGF/p75 in a PC3 prostate cancer cell lysate. WB, Western blotting. C, transcription factor arrays were used to identify candidate interacting transcription factors of LEDGF/p75. Purified His-LEDGF/p75 was incubated with transcription factors spotted on membranes. Protein interactions were detected with human anti-LEDGF/p75 autoantibody and chemiluminescence. A section of the transcription factor array membrane containing MeCP2 is shown.

Article Snippet: Anti-LEDGF/p75 antibodies (A300-848A, Bethyl), antiMeCP2 antibodies (07–013, Millipore), and rabbit IgG (Santa Cruz Biotechnology) were used to immunoprecipitate protein–chromatin complexes.

Techniques: Staining, SDS Page, Transformation Assay, Nickel Column, Western Blot, Incubation, Membrane

Journal: Molecular Cancer Research

Article Title: The Stress Oncoprotein LEDGF/p75 Interacts with the Methyl CpG Binding Protein MeCP2 and Influences Its Transcriptional Activity

doi: 10.1158/1541-7786.mcr-11-0314

Figure Lengend Snippet: Figure 6. The N-terminus of LEDGF/p75 interacts with MeCP2. A, diagram of LEDGF/p75 deletion constructs used to map interaction regions. ATH, AT-hook. CR, charged region. IBD, integrase-binding domain. NLS, nuclear localization signal. B, Flag-MeCP2 binds to eGFP-LEDGF/p75 but not to eGFP-tagged truncated LEDGF/p75 constructs. 293T cells ectopically overexpressing the tagged proteins labeled in the blot were immunoprecipitated (IP) with GFP antibody and visualized by immunoblotting with antibodies to GFP and Flag. C, Flag-MeCP2 binds to eGFP-tagged LEDGF/p75 and LEDGF/p52 but not to truncated constructs. Proteins ectopically overexpressed in U2OS cells were immunoprecipitated with Flag antibody and visualized with both anti-GFP and anti-Flag antibodies. , degradation product of LEDGF/p75. D, recombinant Flag-LEDGF/p75, Flag-PWWP-CR1 (1–141 aa), and Flag-PWWP (1–101 aa) were incubatedwithU2OScelllysate.Proteinspulleddownwithanti-Flagaffinitymatrixweredetectedbyimmunoblotting.EndogenousMeCP2inthecelllysatewas pulled down (PD) by Flag-LEDGF/p75 and Flag-PWWP-CR1. Absence of recombinant proteins (U2OS only) served as negative control. WB, Western blotting.

Article Snippet: Anti-LEDGF/p75 antibodies (A300-848A, Bethyl), antiMeCP2 antibodies (07–013, Millipore), and rabbit IgG (Santa Cruz Biotechnology) were used to immunoprecipitate protein–chromatin complexes.

Techniques: Construct, Binding Assay, Labeling, Immunoprecipitation, Western Blot, Recombinant, Negative Control

Journal: Molecular Cancer Research

Article Title: The Stress Oncoprotein LEDGF/p75 Interacts with the Methyl CpG Binding Protein MeCP2 and Influences Its Transcriptional Activity

doi: 10.1158/1541-7786.mcr-11-0314

Figure Lengend Snippet: Figure 7. LEDGF/p75, LEDGF/p52, and MeCP2 transactivate Hsp27 promoter. U2OS cells were cotransfected with pGL3-Hsp27pr-luc and the indicated amount of (A) empty pcDNA vector or pcDNA-Flag-MeCP2, or (B) pCruzHA, pCruzHA-LEDGF/p75, or pCruzHA-p52 (1 ¼ 1.66 mg DNA). Promoter activity determined as luciferase light units/protein is expressed as fold activation compared with control activity (cotransfection of pGL3-Hsp27pr-luc with empty expression vectors). Data from each graph are representative of at least 3 independent experiments. Representative immunoblots corresponding to the reporter assays show protein expression. C, schematic diagram of Hsp27pr showing AT-rich regions, and HSE and STRE sites. PCR primers targeted Hsp27pr regions A (bp 271 to þ18), B (bp 480 to 220), C (bp 803 to 382), and D (bp 1,071 to 781). D, ChIP analysis of MeCP2 and LEDGF/p75 binding to Hsp27pr. Formaldehyde-fixed chromatin from U2OS cells was precipitated with nonspecific IgG or antibodies specific for MeCP2 or LEDGF/p75. PCR amplifications of immunoprecipitated DNA derived from U2OS cells were carried out with primer sets specific for Hsp27pr regions A to D. Hsp27pr primers amplified DNA fragments precipitated by LEDGF/p75 antibody or MeCP2 antibody but not by IgG. Primers that target human b-actin controlled for optimal enzymatic digestion of chromatin. Data represent the average of at least 3 independent experiments.

Article Snippet: Anti-LEDGF/p75 antibodies (A300-848A, Bethyl), antiMeCP2 antibodies (07–013, Millipore), and rabbit IgG (Santa Cruz Biotechnology) were used to immunoprecipitate protein–chromatin complexes.

Techniques: Plasmid Preparation, Activity Assay, Luciferase, Activation Assay, Control, Cotransfection, Expressing, Western Blot, Binding Assay, Immunoprecipitation, Derivative Assay

Journal: Molecular Cancer Research

Article Title: The Stress Oncoprotein LEDGF/p75 Interacts with the Methyl CpG Binding Protein MeCP2 and Influences Its Transcriptional Activity

doi: 10.1158/1541-7786.mcr-11-0314

Figure Lengend Snippet: Figure 8. LEDGF/p75 and p52 influence MeCP2-induced transactivation of Hsp27pr. A, U2OS cells were cotransfected with pGL3-Hsp27pr-luc, pCruzHA, or pCruzHA-LEDGF/p75, and increasing amounts of pcDNA-Flag-MeCP2 (1 ¼ 1.66 mg DNA). B, U2OS cells were cotransfected with pGL3-Hsp27pr-luc, pCruzHA, or pCruzHA-LEDGF/p52, and increasing amounts of pcDNA-Flag-MeCP2. C, transient knockdown of LEDGF/p75 in U2OS cells was achieved using specific siRNA oligos. Cells were then cotransfected with pcDNA-Flag-MeCP2 and pGL3-Hsp27pr-luc. Promoter activity determined as luciferase light units/protein is expressed as fold activation compared with control activity. D, PC3 cells stably transfected with empty pcDNA vector or pcDNA-LEDGF/p75 were cotransfected with pGL3-Hsp27pr-luc and pMAX-GFP (transfection control). E, PC3 cells stably transfected with empty pcDNA vector or pcDNA-LEDGF/p75 were cotransfected with pGL3-Hsp27pr-luc and pcDNA-Flag-MeCP2. F, PC3 cells stably transfected with empty pcDNA vector or pcDNA-LEDGF/p75 were transfected with siRNA oligos to knockdown this protein. Cells were then cotransfected with pGL3- Hsp27pr-luc and pcDNA-Flag-MeCP2. Promoter activity determined as luciferase light units/GFP is expressed as fold activation compared with control activity. Data represent the average of at least 3 independent experiments. Representative immunoblots corresponding to the reporter assays show protein expression. All protein bands in (F) were from the same blot. , P < 0.05; , P < 0.01.

Article Snippet: Anti-LEDGF/p75 antibodies (A300-848A, Bethyl), antiMeCP2 antibodies (07–013, Millipore), and rabbit IgG (Santa Cruz Biotechnology) were used to immunoprecipitate protein–chromatin complexes.

Techniques: Knockdown, Activity Assay, Luciferase, Activation Assay, Control, Stable Transfection, Transfection, Plasmid Preparation, Western Blot, Expressing

Journal: eLife

Article Title: The Tudor SND1 protein is an m 6 A RNA reader essential for replication of Kaposi’s sarcoma-associated herpesvirus

doi: 10.7554/eLife.47261

Figure Lengend Snippet:

Article Snippet: Antibodies used in RIP are as follows: anti-SND1 (Proteintech, 10760–1-AP); anti-FXR1 (Proteintech, 13194–1-AP); anti-FXR2 (Proteintech, 12552–1-AP); anti-PSIP1 (Proteintech, 25504–1-AP); anti-YTHDF1 (Proteintech, 17479–1-AP); anti-YTHDF3 (Abclonal, A8395) and normal rabbit IgG (Merck Millipore, 12–370).

Techniques: Recombinant, Plasmid Preparation, Expressing, Bacteria, Sequencing, shRNA, Software, Magnetic Beads

Journal: Genome Biology

Article Title: Alternative splicing links histone modifications to stem cell fate decision

doi: 10.1186/s13059-018-1512-3

Figure Lengend Snippet: Isoform switch from PBX1a and PBX1b during hESC differentiation. a Genome browser view shows the AS event and H3K36me3 signals of PBX1 upon hESC differentiation. The green horizontal bars below the ChIP-seq tracks indicate the narrow peaks called by MACS2. b The inclusion level for exon 7 of PBX1 is significantly correlated to the H3K36me3 signals over this exon across cell lineages. c The sequence difference of three protein isoforms of PBX1 and the main functional domains. d The relative expressions of PBX1a and PBX1b in 56 cells/tissues, representing the differential expressions of two isoforms in three groups based on their developmental states. e The expression levels of NANOG and OCT4 genes are negatively correlated with the expression of PBX1b. f The expression levels of PSIP1 and SRSF1 show significant positive correlations with the expression level of PBX1a. Also see Additional file : Figures S9, S10

Article Snippet: The SRSF1-bound PSIP1 protein (also known as LEDGF) level was determined with the PSIP1 antibody (Human LEDGF Antibody, R&D Systems) using western blot as described above.

Techniques: ChIP-sequencing, Sequencing, Functional Assay, Expressing

Journal: Genome Biology

Article Title: Alternative splicing links histone modifications to stem cell fate decision

doi: 10.1186/s13059-018-1512-3

Figure Lengend Snippet: Isoform switch of PBX1 links H3K36me3 to hESC fate decision. a qRT-PCR and western blot show the expression levels of Yamanaka factors in H1, MSC, and IMR90 cells. Whiskers denote the standard deviations of three replicates. b RT-PCR and western blot show the isoform switches between PBX1a and PBX1b from H1 cells to differentiated cells. c i. ChIP-PCR shows the differential binding of PBX1b to NANOG promoter in H1 cells and differentiated cells; ii. ChIP-PCR shows the reduced H3K36me3 signal in differentiated cells; iii. ChIP-PCR shows the differential recruitment of PSIP1 to exon 7 of PBX1. d RIP-PCR show the differential recruitment of SRSF1 around exon 7 of PBX1. e Co-IP shows the overall physical interaction between PSIP1 and SRSF1 in all studied cell types. f The mechanism by which H3K36me3 is linked to cell fate decision by regulating the isoform switch of PBX1, which functions upstream of the pluripotency regulatory network. Also see Additional file : Figures S9, S10

Article Snippet: The SRSF1-bound PSIP1 protein (also known as LEDGF) level was determined with the PSIP1 antibody (Human LEDGF Antibody, R&D Systems) using western blot as described above.

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Reverse Transcription Polymerase Chain Reaction, Binding Assay, Co-Immunoprecipitation Assay

Journal: PloS one

Article Title: Expression of the stress response oncoprotein LEDGF/p75 in human cancer: a study of 21 tumor types.

doi: 10.1371/journal.pone.0030132

Figure Lengend Snippet: Figure 1. Transcript expression of LEDGF/p75 in eight human cancer types determined by TissueScan Cancer Q-PCR analysis. Data were analyzed using the DDCt method with values normalized to b-actin levels. The y-axis represents the induction fold of the LEDGF/p75 mRNA level in eight cancer types (n = 9) compared to matching normal adjacent tissues (n = 3) in the array. Error bars displays the range of standard error. * P,0.05. P values were determined with Student’s t-test. doi:10.1371/journal.pone.0030132.g001

Article Snippet: Antibodies and Immunoblotting The following antibodies were used: mouse monoclonals anti-bactin (Sigma-Aldrich), and anti-LEDGF (1:1000, BD Biosciences); rabbit polyclonals anti-LEDGF/p75 (1:1000, Novus Biologicals); anti-LEDGF/p75 (1:1000, Bethyl); and horseradish peroxidase (HRP)-labeled secondary IgG antibodies (Zymed).

Techniques: Expressing

Journal: PloS one

Article Title: Expression of the stress response oncoprotein LEDGF/p75 in human cancer: a study of 21 tumor types.

doi: 10.1371/journal.pone.0030132

Figure Lengend Snippet: Figure 2. Identification of LEDGF/p75 specific antibody by immunoblotting in PC-3 cells with transient LEDGF/p75 knockdown. Cells were transfected with siLEDGF/p75 to induce transient knockdown of LEDGF/p75. PC-3 cells transfected with small interfering scrambled RNA duplex (siSD) served as corresponding control. Immunoblotting analysis tested the specific reactivity of all the antibodies against LEDGF/PSIP1. All the blot pairs (siSD and siLEDGF/p75) for each antibody were derived from the same blot. doi:10.1371/journal.pone.0030132.g002

Article Snippet: Antibodies and Immunoblotting The following antibodies were used: mouse monoclonals anti-bactin (Sigma-Aldrich), and anti-LEDGF (1:1000, BD Biosciences); rabbit polyclonals anti-LEDGF/p75 (1:1000, Novus Biologicals); anti-LEDGF/p75 (1:1000, Bethyl); and horseradish peroxidase (HRP)-labeled secondary IgG antibodies (Zymed).

Techniques: Western Blot, Knockdown, Transfection, Control, Derivative Assay

Journal: PloS one

Article Title: Expression of the stress response oncoprotein LEDGF/p75 in human cancer: a study of 21 tumor types.

doi: 10.1371/journal.pone.0030132

Figure Lengend Snippet: Figure 3. Elevated immunohistochemical expression of LEDGF/p75 protein in five tumor types compared to corresponding normal tissues. Tissue microarrays were stained with antibody against LEDGF/p75, and the individual cores were blindly scored using the following scale: 0 = no staining, 1 = low staining, 2 = moderate staining, 3 = strong staining. Scored tissues were pooled into two groups: low staining (scores 0 and 1, dark bars) and high staining (scores 2 and 3, light bars). The percentage of specimens in the two staining categories was plotted for tumor tissues compared to normal (including disease-free normal and normal adjacent) tissues. *P,0.05; **P,0.01. P values were determined with Fisher’s exact test. doi:10.1371/journal.pone.0030132.g003

Article Snippet: Antibodies and Immunoblotting The following antibodies were used: mouse monoclonals anti-bactin (Sigma-Aldrich), and anti-LEDGF (1:1000, BD Biosciences); rabbit polyclonals anti-LEDGF/p75 (1:1000, Novus Biologicals); anti-LEDGF/p75 (1:1000, Bethyl); and horseradish peroxidase (HRP)-labeled secondary IgG antibodies (Zymed).

Techniques: Immunohistochemical staining, Expressing, Staining

Journal: PloS one

Article Title: Expression of the stress response oncoprotein LEDGF/p75 in human cancer: a study of 21 tumor types.

doi: 10.1371/journal.pone.0030132

Figure Lengend Snippet: Figure 4. Immunohistochemical staining for LEDGF/p75 protein in selected human tumors. A. Representative images of immunohistochemical staining (low intensity, scores 0–1; high intensity, scores 2–3) for LEDGF/p75 in prostate, colon, and thyroid tumors (Scale bar-40 mm; magnification = 2006). B. Representative images of LEDGF/p75 immunostaining of non-disease normal prostate and colon tissues, and in tumor tissues and their matched adjacent tissues (Scale bar-40 mm; magnification = 4006). TMAs were stained using LEDGF/p75 specific rabbit antibody Scripps-Ab5087, as indicated in Materials and Methods. Identical camera settings were used in the acquisition and processing of images for a particular tissue type. doi:10.1371/journal.pone.0030132.g004

Article Snippet: Antibodies and Immunoblotting The following antibodies were used: mouse monoclonals anti-bactin (Sigma-Aldrich), and anti-LEDGF (1:1000, BD Biosciences); rabbit polyclonals anti-LEDGF/p75 (1:1000, Novus Biologicals); anti-LEDGF/p75 (1:1000, Bethyl); and horseradish peroxidase (HRP)-labeled secondary IgG antibodies (Zymed).

Techniques: Immunohistochemical staining, Staining, Immunostaining

Journal: Journal of Molecular Cell Biology

Article Title: HIV-induced membraneless organelles orchestrate post-nuclear entry steps

doi: 10.1093/jmcb/mjac060

Figure Lengend Snippet: HIV-1 active proviruses locate in LEDGF-marked chromatin neighboring HIV-1 MLOs. ( A ) Confocal images of THP-1 cells expressing OR-GFP infected with HIV-1 ANCH3 (MOI = 10, 3 days p.i.) and expressing MCP-GFP infected with HIV-1 ANCH3 MS2 (MOI = 2.5, 3 days p.i.). On the bottom, the plots show the percentage of CPSF6 clusters associated with LEDGF per nucleus ± SD (38 cells, 161 CPSF6) and the percentage of vDNA or vRNA associated with LEDGF cluster per nucleus ± SD. Two independent experiments were performed. ( B ) Scatter plot shows the distance of vDNA or vRNA from the border of the closest HIV-1 MLO (SC35 + CPSF6) ± SD (75 vDNAs, 7 cells; 34 vRNAs, 8 cells). Unpaired t -test, ** P ≤ 0.01. Results of two biological replicates were analyzed from 3D acquisitions. Scale bar, 5 µm. Inset, 1 µm.

Article Snippet: Primary antibodies were diluted as follows: anti-HA 1:500 (Roche #11867423001), anti-CPSF6 1:400 (Novus Biologicals #NBP1-85676), anti-SC35 1:200 (Abcam #ab11826), and anti-LEDGF 1:200 (BD Bioscience #611715).

Techniques: Expressing, Infection

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