anti flna  (Bethyl)

 
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 88
    Name:
    Rabbit anti THRAP1 TRAP240 Antibody Affinity Purified
    Description:

    Catalog Number:
    A301-277A
    Price:
    [339.0]
    Applications:
    Western Blot,Immunoprecipitation
    Host:
    Rabbit
    Conjugate:
    Unconjugated
    Size:
    20 ug
    Category:
    Antibody
    Antibody Type:
    Primary antibody
    Isotype:
    IgG
    Reactivity:
    Human
    Buy from Supplier


    Structured Review

    Bethyl anti flna
    Spatiotemporal activation of Rap1 reduces association of FLN with β 2 . ( a ) RalGDS-RBD mCherry in control BAF cells stimulated with CXCL12 as indicated. Scale bar, 5 μm. ( b ) FRET-based Rap1 activity sensor-expressing control BAF cells stimulated with CXCL12 as indicated. A ratio image of mTurquoise/CFP represents FRET efficiency. Scale bar, 5 μm. ( c ) Control cells expressing RalGDS-RBD mCherry perfused on the endothelium with CXCL12. The digital images of contact areas of the cells taken under shear flow. Scale bar, 5 μm. ( d ) COS cells transfected with T7-Rap1V12, Rap1N17 or αL and β 2 were lysed and pulled down using GST fusions of <t>FLNa</t> repeats 1–3, 4–6, 7–10 or 21, and immunoblotted with anti-T7 or anti-β 2 . ( e ; Upper) Lysates from COS cells transfected with T7-Rap1V12 or Rap1N17 immunoprecipitated with anti-FLNa, and immunoblotted with anti-T7. (Lower) Lysates from Cos cells transfected with Halo-FLNa wild-type and the mutants deleting repeat 1(Δ1), 2 (Δ2) or 3(Δ3), and T7-Rap1V12, precipitated using HaloLink Resin, and immunoblotted with anti-T7 or Halo. ( f ) Lysates from COS cells transfected with Halo-FLNa, T7-Rap1V12 or N17, and αL and mRFP-β precipitated with HaloLink resin, and immunoblotted with anti-mRFP, T7, Halo and αL. ( g ) Membrane fraction from BAF/LFA-1 cells transfected with vector or T7-Rap1V12 pulled down using the cytoplasmic region of β 2 -GST fusion protein. Bound FLNa and total FLNa and T7-Rap1V12 were analysed using anti-FLNa or anti-T7 antibody. ( h ) Lysates from BAF/LFA-1 cells transfected with vector or Spa-1 , ±CXCL12 for 1 min, immunoprecipitated with anti-β 2 , and immunoblotted with anti-FLNa. ( i ; Left upper) Western blots of total cell lysates from BAF/LFA-1/L-selectin cells transduced with scramble (Control) or FLNa or b -targeting shRNA. Actin is a loading control. (Left lower) Epitope expression of monoclonal antibody KIM127. Data are normalized against LFA-1 expression detected by <t>TS1/18.</t> * P

    https://www.bioz.com/result/anti flna/product/Bethyl
    Average 88 stars, based on 116 article reviews
    Price from $9.99 to $1999.99
    anti flna - by Bioz Stars, 2020-09
    88/100 stars

    Images

    1) Product Images from "Dual functions of Rap1 are crucial for T-cell homeostasis and prevention of spontaneous colitis"

    Article Title: Dual functions of Rap1 are crucial for T-cell homeostasis and prevention of spontaneous colitis

    Journal: Nature Communications

    doi: 10.1038/ncomms9982

    Spatiotemporal activation of Rap1 reduces association of FLN with β 2 . ( a ) RalGDS-RBD mCherry in control BAF cells stimulated with CXCL12 as indicated. Scale bar, 5 μm. ( b ) FRET-based Rap1 activity sensor-expressing control BAF cells stimulated with CXCL12 as indicated. A ratio image of mTurquoise/CFP represents FRET efficiency. Scale bar, 5 μm. ( c ) Control cells expressing RalGDS-RBD mCherry perfused on the endothelium with CXCL12. The digital images of contact areas of the cells taken under shear flow. Scale bar, 5 μm. ( d ) COS cells transfected with T7-Rap1V12, Rap1N17 or αL and β 2 were lysed and pulled down using GST fusions of FLNa repeats 1–3, 4–6, 7–10 or 21, and immunoblotted with anti-T7 or anti-β 2 . ( e ; Upper) Lysates from COS cells transfected with T7-Rap1V12 or Rap1N17 immunoprecipitated with anti-FLNa, and immunoblotted with anti-T7. (Lower) Lysates from Cos cells transfected with Halo-FLNa wild-type and the mutants deleting repeat 1(Δ1), 2 (Δ2) or 3(Δ3), and T7-Rap1V12, precipitated using HaloLink Resin, and immunoblotted with anti-T7 or Halo. ( f ) Lysates from COS cells transfected with Halo-FLNa, T7-Rap1V12 or N17, and αL and mRFP-β precipitated with HaloLink resin, and immunoblotted with anti-mRFP, T7, Halo and αL. ( g ) Membrane fraction from BAF/LFA-1 cells transfected with vector or T7-Rap1V12 pulled down using the cytoplasmic region of β 2 -GST fusion protein. Bound FLNa and total FLNa and T7-Rap1V12 were analysed using anti-FLNa or anti-T7 antibody. ( h ) Lysates from BAF/LFA-1 cells transfected with vector or Spa-1 , ±CXCL12 for 1 min, immunoprecipitated with anti-β 2 , and immunoblotted with anti-FLNa. ( i ; Left upper) Western blots of total cell lysates from BAF/LFA-1/L-selectin cells transduced with scramble (Control) or FLNa or b -targeting shRNA. Actin is a loading control. (Left lower) Epitope expression of monoclonal antibody KIM127. Data are normalized against LFA-1 expression detected by TS1/18. * P
    Figure Legend Snippet: Spatiotemporal activation of Rap1 reduces association of FLN with β 2 . ( a ) RalGDS-RBD mCherry in control BAF cells stimulated with CXCL12 as indicated. Scale bar, 5 μm. ( b ) FRET-based Rap1 activity sensor-expressing control BAF cells stimulated with CXCL12 as indicated. A ratio image of mTurquoise/CFP represents FRET efficiency. Scale bar, 5 μm. ( c ) Control cells expressing RalGDS-RBD mCherry perfused on the endothelium with CXCL12. The digital images of contact areas of the cells taken under shear flow. Scale bar, 5 μm. ( d ) COS cells transfected with T7-Rap1V12, Rap1N17 or αL and β 2 were lysed and pulled down using GST fusions of FLNa repeats 1–3, 4–6, 7–10 or 21, and immunoblotted with anti-T7 or anti-β 2 . ( e ; Upper) Lysates from COS cells transfected with T7-Rap1V12 or Rap1N17 immunoprecipitated with anti-FLNa, and immunoblotted with anti-T7. (Lower) Lysates from Cos cells transfected with Halo-FLNa wild-type and the mutants deleting repeat 1(Δ1), 2 (Δ2) or 3(Δ3), and T7-Rap1V12, precipitated using HaloLink Resin, and immunoblotted with anti-T7 or Halo. ( f ) Lysates from COS cells transfected with Halo-FLNa, T7-Rap1V12 or N17, and αL and mRFP-β precipitated with HaloLink resin, and immunoblotted with anti-mRFP, T7, Halo and αL. ( g ) Membrane fraction from BAF/LFA-1 cells transfected with vector or T7-Rap1V12 pulled down using the cytoplasmic region of β 2 -GST fusion protein. Bound FLNa and total FLNa and T7-Rap1V12 were analysed using anti-FLNa or anti-T7 antibody. ( h ) Lysates from BAF/LFA-1 cells transfected with vector or Spa-1 , ±CXCL12 for 1 min, immunoprecipitated with anti-β 2 , and immunoblotted with anti-FLNa. ( i ; Left upper) Western blots of total cell lysates from BAF/LFA-1/L-selectin cells transduced with scramble (Control) or FLNa or b -targeting shRNA. Actin is a loading control. (Left lower) Epitope expression of monoclonal antibody KIM127. Data are normalized against LFA-1 expression detected by TS1/18. * P

    Techniques Used: Activation Assay, Activity Assay, Expressing, Flow Cytometry, Transfection, Immunoprecipitation, Plasmid Preparation, Western Blot, Transduction, shRNA

    2) Product Images from "Integrated genomic approaches identify upregulation of SCRN1 as a novel mechanism associated with acquired resistance to erlotinib in PC9 cells harboring oncogenic EGFR mutation"

    Article Title: Integrated genomic approaches identify upregulation of SCRN1 as a novel mechanism associated with acquired resistance to erlotinib in PC9 cells harboring oncogenic EGFR mutation

    Journal: Oncotarget

    doi: 10.18632/oncotarget.7318

    Downregulation of SCRN1 in erlotinib-resistant cell clones enhanced the drug sensitivity and cellular apoptosis in response to erlotinib ( A ) Suppression of SCRN1 by shRNA in erlotinib resistant clones increased erlotinib sensitivity. A549 cells were used as a negative control for the experiment. ( B ) Resistant clone C1 and C2 respond to erlotinib following SCRN1 knockdown by shRNA in concentration dependent manner. The results are indicated as mean +/− SD of sextuplicate wells and are representative of three independent experiments. ( C ) C1 and C2 resistant clones are dependent on SCRN1 for their transforming potential. The bar graph depicts the relative number of colonies in C1 or C2 transfected with sh SCRN1 normalized to the number of colonies formed by cells transfected with shGFP ( n = 3, mean + SD). ( D ) Knockdown of SCRN1 increases caspase 3/7 activities in C1 and C2 clones. Values are the means + SD from three independent experiments.
    Figure Legend Snippet: Downregulation of SCRN1 in erlotinib-resistant cell clones enhanced the drug sensitivity and cellular apoptosis in response to erlotinib ( A ) Suppression of SCRN1 by shRNA in erlotinib resistant clones increased erlotinib sensitivity. A549 cells were used as a negative control for the experiment. ( B ) Resistant clone C1 and C2 respond to erlotinib following SCRN1 knockdown by shRNA in concentration dependent manner. The results are indicated as mean +/− SD of sextuplicate wells and are representative of three independent experiments. ( C ) C1 and C2 resistant clones are dependent on SCRN1 for their transforming potential. The bar graph depicts the relative number of colonies in C1 or C2 transfected with sh SCRN1 normalized to the number of colonies formed by cells transfected with shGFP ( n = 3, mean + SD). ( D ) Knockdown of SCRN1 increases caspase 3/7 activities in C1 and C2 clones. Values are the means + SD from three independent experiments.

    Techniques Used: Clone Assay, shRNA, Negative Control, Concentration Assay, Transfection

    Increased SCRN1 levels were detected in a subset of patient specimens from EGFR-TKIs resistant lung adenocarcinoma patients ( A ) Schematic summary of 11 primary tumor specimens obtained from patients with acquired EGFR-TKI resistant lung adenocarcinoma for the status on T790M mutation in EGFR and SCRN1 protein expression determined by immunohistochemistry. ( B ) Immunohistochemical staining for SCRN1. Representative images from specimens (patient 2 and patient 8) that show negative and positive SCRN1 immunohistochemical staining, respectively.
    Figure Legend Snippet: Increased SCRN1 levels were detected in a subset of patient specimens from EGFR-TKIs resistant lung adenocarcinoma patients ( A ) Schematic summary of 11 primary tumor specimens obtained from patients with acquired EGFR-TKI resistant lung adenocarcinoma for the status on T790M mutation in EGFR and SCRN1 protein expression determined by immunohistochemistry. ( B ) Immunohistochemical staining for SCRN1. Representative images from specimens (patient 2 and patient 8) that show negative and positive SCRN1 immunohistochemical staining, respectively.

    Techniques Used: Mutagenesis, Expressing, Immunohistochemistry, Staining

    Identification of SCRN1 upregulation as a potential erlotinib resistant gene by RNAseq analysis followed by siRNA synthetic lethality screening ( A ) Growth of five isolated resistant PC9/CYF10 cell clones (C1–C5) is unaffected with erlotinib treatment. The results are presented as a mean ± SD of sextuplicate wells and are representative of three independent experiments. ( B ) Xenograft of erlotinib resistant clones generate tumor and remain refractory to erlotinib treatment. ( C ) Rank-ordered by statistical SAM score for differential expression in erlotinib-resistant PC9/CYF10 cells compared to erlotinib-sensitive parental cells from RNA-seq data and plotted against expected SAM score. A total of 21,514 genes are plotted. Red circles indicate significantly upregulated genes ( n = 80) in erlotinib resistant cells with a score that deviates from expected distribution at delta slope of 2.5. Green circles indicate significantly downregulated genes ( n = 47) in erlotinib resistant cell lines. ( D ) Schematic of siRNA synthetic lethality loss-of-function screen measuring cell viability in the presence or absence of the erlotinib. ( E ) Overlapping hits selected from data analysis of siRNA screening in three conditions are shown in the Venn diagram. The 10 overlapping genes among the three conditions are listed in the Figure ( F ) The levels of SCRN1 protein are significantly elevated in all erlotinib-resistant clones compared to parental control cells as shown by immunoblot analysis. Vinculin serves as a loading control. ( G ) Quantitative RT-PCR for SCRN1 in parental and resistant clones validated that mRNA levels of SCRN1 clones are higher in C1 and C2 than parental control cells. The fold change in SCRN1 expression is shown in log2 in graph.
    Figure Legend Snippet: Identification of SCRN1 upregulation as a potential erlotinib resistant gene by RNAseq analysis followed by siRNA synthetic lethality screening ( A ) Growth of five isolated resistant PC9/CYF10 cell clones (C1–C5) is unaffected with erlotinib treatment. The results are presented as a mean ± SD of sextuplicate wells and are representative of three independent experiments. ( B ) Xenograft of erlotinib resistant clones generate tumor and remain refractory to erlotinib treatment. ( C ) Rank-ordered by statistical SAM score for differential expression in erlotinib-resistant PC9/CYF10 cells compared to erlotinib-sensitive parental cells from RNA-seq data and plotted against expected SAM score. A total of 21,514 genes are plotted. Red circles indicate significantly upregulated genes ( n = 80) in erlotinib resistant cells with a score that deviates from expected distribution at delta slope of 2.5. Green circles indicate significantly downregulated genes ( n = 47) in erlotinib resistant cell lines. ( D ) Schematic of siRNA synthetic lethality loss-of-function screen measuring cell viability in the presence or absence of the erlotinib. ( E ) Overlapping hits selected from data analysis of siRNA screening in three conditions are shown in the Venn diagram. The 10 overlapping genes among the three conditions are listed in the Figure ( F ) The levels of SCRN1 protein are significantly elevated in all erlotinib-resistant clones compared to parental control cells as shown by immunoblot analysis. Vinculin serves as a loading control. ( G ) Quantitative RT-PCR for SCRN1 in parental and resistant clones validated that mRNA levels of SCRN1 clones are higher in C1 and C2 than parental control cells. The fold change in SCRN1 expression is shown in log2 in graph.

    Techniques Used: Isolation, Clone Assay, Expressing, RNA Sequencing Assay, Quantitative RT-PCR

    Activation of PI3K/AKT signaling pathways is essential for growth of erlotinib resistant cells ( A ) Caspase 3/7 activities in C1 and C2 cells treated with EGFR-TKIs were significantly enhanced following SCRN1 knockdown compared to sh GFP control. Values are the means + SD from three independent experiments. ( B ) Levels of constitutively phosphorylated AKT and ERK1/2 were more robustly reduced by either erlotinib or dacomitinib in C1 cells transfected with shSCRN1 than in those with sh GFP . ( C ) Growth of C1 and C2 cells in presence of PI3K/AKT inhibitor NVP-BEZ235 is equivalent to that of PC9 parental cells. The results are presented as a mean ± SD of sextuplicate wells and are representative of three independent experiments. ( D and E ) Erlotinib synergistically increased the sensitivity of NVP-BEZ235 for PC9 cell (C), but not for C1 cells (D). The results are presented as a mean ± SD of sextuplicate wells and are representative of three independent experiments. ( F ) Growth of C1 cells was synergistically inhibited by NVP-BEZ235 in combination with shRNA-mediated silencing of SCRN1. The results are presented as a mean ± SD of sextuplicate wells and are representative of three independent experiments.
    Figure Legend Snippet: Activation of PI3K/AKT signaling pathways is essential for growth of erlotinib resistant cells ( A ) Caspase 3/7 activities in C1 and C2 cells treated with EGFR-TKIs were significantly enhanced following SCRN1 knockdown compared to sh GFP control. Values are the means + SD from three independent experiments. ( B ) Levels of constitutively phosphorylated AKT and ERK1/2 were more robustly reduced by either erlotinib or dacomitinib in C1 cells transfected with shSCRN1 than in those with sh GFP . ( C ) Growth of C1 and C2 cells in presence of PI3K/AKT inhibitor NVP-BEZ235 is equivalent to that of PC9 parental cells. The results are presented as a mean ± SD of sextuplicate wells and are representative of three independent experiments. ( D and E ) Erlotinib synergistically increased the sensitivity of NVP-BEZ235 for PC9 cell (C), but not for C1 cells (D). The results are presented as a mean ± SD of sextuplicate wells and are representative of three independent experiments. ( F ) Growth of C1 cells was synergistically inhibited by NVP-BEZ235 in combination with shRNA-mediated silencing of SCRN1. The results are presented as a mean ± SD of sextuplicate wells and are representative of three independent experiments.

    Techniques Used: Activation Assay, Transfection, shRNA

    Silencing of SCRN1 by shRNA significantly increased apoptosis induced by EGFR TKIs in T790M-bearing NCI-H1975 cells ( A ) Levels of SCRN1 protein in various lung adenocarcinoma cell lines were examined by immunoblotting analysis. ( B ) Caspase 3/7 activities induced by EGFR-TKIs in NCI-H1975 cells, but not in A549 cells, were significantly enhanced following SCRN1 knockdown compared to sh GFP control. Values are the means + SD from three independent experiments. ( C ) Levels of phospho-AKT were synergistically diminished by EGFR-TKIs treatment in NCI-H1975 cells expressing shSCRN1. ( D ) Gwoth of H1975 cells are synergistically inhibited by treatment of NVP-BEZ235 and/or erlotinib following shSCRN1 transfection.
    Figure Legend Snippet: Silencing of SCRN1 by shRNA significantly increased apoptosis induced by EGFR TKIs in T790M-bearing NCI-H1975 cells ( A ) Levels of SCRN1 protein in various lung adenocarcinoma cell lines were examined by immunoblotting analysis. ( B ) Caspase 3/7 activities induced by EGFR-TKIs in NCI-H1975 cells, but not in A549 cells, were significantly enhanced following SCRN1 knockdown compared to sh GFP control. Values are the means + SD from three independent experiments. ( C ) Levels of phospho-AKT were synergistically diminished by EGFR-TKIs treatment in NCI-H1975 cells expressing shSCRN1. ( D ) Gwoth of H1975 cells are synergistically inhibited by treatment of NVP-BEZ235 and/or erlotinib following shSCRN1 transfection.

    Techniques Used: shRNA, Expressing, Transfection

    3) Product Images from "NR2F1 disrupts synergistic activation of the MTTP gene transcription by HNF-4? and HNF-1?"

    Article Title: NR2F1 disrupts synergistic activation of the MTTP gene transcription by HNF-4? and HNF-1?

    Journal: Journal of Lipid Research

    doi: 10.1194/jlr.M025130

    Identification of the cis elements required for synergistic activation of the MTTP promoter by HNF-4α and HNF-1α. A: Left, schematic diagram showing location of three different cis elements in the WT promoter. Right, cotransfection of
    Figure Legend Snippet: Identification of the cis elements required for synergistic activation of the MTTP promoter by HNF-4α and HNF-1α. A: Left, schematic diagram showing location of three different cis elements in the WT promoter. Right, cotransfection of

    Techniques Used: Activation Assay, Cotransfection

    Synergistic activation potential of HNF-1 family proteins with HNF-4α. A: Expression of three different HNF-1 proteins, HNF-1α, HNF-1β(a), and HNF-1β(b), in HEK293 cells was determined by Western blotting using specific
    Figure Legend Snippet: Synergistic activation potential of HNF-1 family proteins with HNF-4α. A: Expression of three different HNF-1 proteins, HNF-1α, HNF-1β(a), and HNF-1β(b), in HEK293 cells was determined by Western blotting using specific

    Techniques Used: Activation Assay, Expressing, Western Blot

    NR2F1 binds to the DR1 element and represses synergistic activation of MTTP promoter by HNF-4α/HNF-1α. A: WT and mutant pMTP-204 plasmids as illustrated were cotransfected with HNF-4α/HNF-1α expression vectors along with
    Figure Legend Snippet: NR2F1 binds to the DR1 element and represses synergistic activation of MTTP promoter by HNF-4α/HNF-1α. A: WT and mutant pMTP-204 plasmids as illustrated were cotransfected with HNF-4α/HNF-1α expression vectors along with

    Techniques Used: Activation Assay, Mutagenesis, Expressing

    Mechanisms involved in the repression of the MTTP promoter by NR2F1. A: Western blot analysis showing overexpression of NR2F1 in HEK293 cells B: NR2F1 does not affect the activation potential of HNF-1α. Cells were transfected with different combinations
    Figure Legend Snippet: Mechanisms involved in the repression of the MTTP promoter by NR2F1. A: Western blot analysis showing overexpression of NR2F1 in HEK293 cells B: NR2F1 does not affect the activation potential of HNF-1α. Cells were transfected with different combinations

    Techniques Used: Western Blot, Over Expression, Activation Assay, Transfection

    4) Product Images from "Cytoplasmic ATXN7L3B Interferes with Nuclear Functions of the SAGA Deubiquitinase Module"

    Article Title: Cytoplasmic ATXN7L3B Interferes with Nuclear Functions of the SAGA Deubiquitinase Module

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00193-16

    ATXN7L3B mainly localizes to the cytoplasm, whereas ATXN7L3 is nuclear. (A) Immunofluorescence using Flag antibody (red) on 293T cells stably expressing pINTO-N-FH vector, pINTO-N-FH-ATXN7L3, or pINTO-N-FH-ATXN7L3B. Nuclei were counterstained with DAPI
    Figure Legend Snippet: ATXN7L3B mainly localizes to the cytoplasm, whereas ATXN7L3 is nuclear. (A) Immunofluorescence using Flag antibody (red) on 293T cells stably expressing pINTO-N-FH vector, pINTO-N-FH-ATXN7L3, or pINTO-N-FH-ATXN7L3B. Nuclei were counterstained with DAPI

    Techniques Used: Immunofluorescence, Stable Transfection, Expressing, Plasmid Preparation

    ATXN7L3 and ATXN7L3B affect global levels and subcellular distributions of H2Bub1, ENY2, and USP22. (A) Whole-cell lysates from 293T cells stably expressing pINTO-N-FH vector, pINTO-N-FH-ATXN7L3, or pINTO-N-FH-ATXN7L3B were resolved by SDS-PAGE. Proteins
    Figure Legend Snippet: ATXN7L3 and ATXN7L3B affect global levels and subcellular distributions of H2Bub1, ENY2, and USP22. (A) Whole-cell lysates from 293T cells stably expressing pINTO-N-FH vector, pINTO-N-FH-ATXN7L3, or pINTO-N-FH-ATXN7L3B were resolved by SDS-PAGE. Proteins

    Techniques Used: Stable Transfection, Expressing, Plasmid Preparation, SDS Page

    Depletion of ATXN7L3B inhibits migration of ER-positive breast cancer cells. (A) Protein levels of ATXN7L3B in two normal mammary, four ER + , and three HER2 + cell lines were detected by immunoblotting. (B and C) Efficient silencing of ATXN7L3B in MCF7T
    Figure Legend Snippet: Depletion of ATXN7L3B inhibits migration of ER-positive breast cancer cells. (A) Protein levels of ATXN7L3B in two normal mammary, four ER + , and three HER2 + cell lines were detected by immunoblotting. (B and C) Efficient silencing of ATXN7L3B in MCF7T

    Techniques Used: Migration

    ATXN7L3B competes with ATXN7L3 for ENY2 binding in vitro . (A) Recombinant proteins were purified from Sf21 cells and resolved by electrophoresis, followed by colloidal blue staining. Asterisks indicate background protein bands. (B) The indicated recombinant
    Figure Legend Snippet: ATXN7L3B competes with ATXN7L3 for ENY2 binding in vitro . (A) Recombinant proteins were purified from Sf21 cells and resolved by electrophoresis, followed by colloidal blue staining. Asterisks indicate background protein bands. (B) The indicated recombinant

    Techniques Used: Binding Assay, In Vitro, Recombinant, Purification, Electrophoresis, Staining

    ATXN7L3B interacts with DUB module components but not SAGA. (A) Comparison of the protein structures of ATXN7L3 and ATXN7L3B. The N termini share 74% identity, but ATXN7L3B lacks the Sgf11 and SCA7 domains that are present in ATXN7L3. (B) Comparison of
    Figure Legend Snippet: ATXN7L3B interacts with DUB module components but not SAGA. (A) Comparison of the protein structures of ATXN7L3 and ATXN7L3B. The N termini share 74% identity, but ATXN7L3B lacks the Sgf11 and SCA7 domains that are present in ATXN7L3. (B) Comparison of

    Techniques Used:

    Regulation of protein levels of H2Bub1 and DUB components by ATXN7L3 and ATXN7L3B. ATXN7L3 mainly resides in the nucleus, while ATXN7L3B mainly localizes in the cytoplasm. They both interact strongly with ENY2. Overexpression of ATXN7L3 leads to increases
    Figure Legend Snippet: Regulation of protein levels of H2Bub1 and DUB components by ATXN7L3 and ATXN7L3B. ATXN7L3 mainly resides in the nucleus, while ATXN7L3B mainly localizes in the cytoplasm. They both interact strongly with ENY2. Overexpression of ATXN7L3 leads to increases

    Techniques Used: Over Expression

    ATXN7L3B regulates the protein levels of ENY2. (A) Whole-cell lysates from MCF7T cells stably expressing shATXN7L3, shATXN7L3B, or nontargeting shRNA (pGIPZ) were resolved by SDS-PAGE. Proteins were transferred onto membranes and detected by immunoblotting
    Figure Legend Snippet: ATXN7L3B regulates the protein levels of ENY2. (A) Whole-cell lysates from MCF7T cells stably expressing shATXN7L3, shATXN7L3B, or nontargeting shRNA (pGIPZ) were resolved by SDS-PAGE. Proteins were transferred onto membranes and detected by immunoblotting

    Techniques Used: Stable Transfection, Expressing, shRNA, SDS Page

    5) Product Images from "NOD2 deficiency exacerbates hypoxia-induced pulmonary hypertension and enhances pulmonary vascular smooth muscle cell proliferation"

    Article Title: NOD2 deficiency exacerbates hypoxia-induced pulmonary hypertension and enhances pulmonary vascular smooth muscle cell proliferation

    Journal: Oncotarget

    doi: 10.18632/oncotarget.23912

    Absence of NOD2 enhances the stability of HIF-1α protein in PASMCs exposed to hypoxic conditions ( A ) Total protein was extracted from NOD2 +/+ and NOD2 −/− PASMCs exposed to normoxic (N) or hypoxic conditions for the indicated lengths of time. The protein levels of HIF-1α, hydroxylated HIF-1α (Pro564), HIF-1β, HIF-2α, PHD2 and VHL were then assessed by Western blot; β-actin was used as a loading control. Experiments were performed at least three independent times. ( B – D ), Total RNA was extracted from NOD2 +/+ and NOD2 −/− PASMCs exposed to normoxic (N) or hypoxic conditions for the indicated lengths of time. The mRNA levels of Hif-1α (B), Hif-1 β (C) and Hif-2α (D) were then analyzed by quantitative real-time RT-PCR; mouse β -actin was used as a control for normalization. * P
    Figure Legend Snippet: Absence of NOD2 enhances the stability of HIF-1α protein in PASMCs exposed to hypoxic conditions ( A ) Total protein was extracted from NOD2 +/+ and NOD2 −/− PASMCs exposed to normoxic (N) or hypoxic conditions for the indicated lengths of time. The protein levels of HIF-1α, hydroxylated HIF-1α (Pro564), HIF-1β, HIF-2α, PHD2 and VHL were then assessed by Western blot; β-actin was used as a loading control. Experiments were performed at least three independent times. ( B – D ), Total RNA was extracted from NOD2 +/+ and NOD2 −/− PASMCs exposed to normoxic (N) or hypoxic conditions for the indicated lengths of time. The mRNA levels of Hif-1α (B), Hif-1 β (C) and Hif-2α (D) were then analyzed by quantitative real-time RT-PCR; mouse β -actin was used as a control for normalization. * P

    Techniques Used: Western Blot, Quantitative RT-PCR

    6) Product Images from "Cyclin C is a haploinsufficient tumor suppressor"

    Article Title: Cyclin C is a haploinsufficient tumor suppressor

    Journal: Nature cell biology

    doi: 10.1038/ncb3046

    Gene expression analyses of cyclin C-null cells. ( a ) CDK8 was immunoprecipitated (IP) from wild-type (WT) or cyclin C Δ/Δ (C-KO) MEFs and used for in vitro kinase reactions with recombinant carboxy terminal domain (CTD) of RNA polymerase II as a substrate, in the presence of γ[ 32 P]ATP. C-KO + cyclin C: cyclin C Δ/Δ MEFs engineered to ectopically express cyclin C. Note that re-expression of cyclin C restored CDK8 kinase activity in cyclin C Δ/Δ cells. IgG was used for control immunoprecipitation. 32 P-CTD denotes phosphorylated CTD, detected by autoradiography. Lower panel, CTD was visualized by Coomassie staining. ( b ) Lysates from wild-type (WT), cyclin C +/Δ (C-HET) and cyclin C Δ/Δ (C-KO) ES cells were probed with an antibody against phospho-Ser 2 and phospho-Ser 5 of RNA polymerase II CTD. Lower panel: immunoblotting with an anti-CTD antibody. ( c ) Cyclin C, CDK8, MED12 or MED13 were immunoprecipitated (IP) from wild-type or C-KO MEFs; the immunoblots were probed with the indicated antibodies, along with whole cell extracts (Input). ( d ) Scatterplot showing log of the normalized gene expression in control (x-axis) vs. C Δ/Δ (y-axis) MEFs, ES cells (ESC) and E18.5 brains. Parallel lines indicate 2-fold change (up- or down-regulation) in transcript levels. ( e ) Scatterplot showing log of the normalized gene expression values in control brains (x-axis) vs. control ES cells (y-axis). This is a control to panel d . It illustrates that different compartments (brain, ES cells) display very distinct gene expression patterns, as expected.
    Figure Legend Snippet: Gene expression analyses of cyclin C-null cells. ( a ) CDK8 was immunoprecipitated (IP) from wild-type (WT) or cyclin C Δ/Δ (C-KO) MEFs and used for in vitro kinase reactions with recombinant carboxy terminal domain (CTD) of RNA polymerase II as a substrate, in the presence of γ[ 32 P]ATP. C-KO + cyclin C: cyclin C Δ/Δ MEFs engineered to ectopically express cyclin C. Note that re-expression of cyclin C restored CDK8 kinase activity in cyclin C Δ/Δ cells. IgG was used for control immunoprecipitation. 32 P-CTD denotes phosphorylated CTD, detected by autoradiography. Lower panel, CTD was visualized by Coomassie staining. ( b ) Lysates from wild-type (WT), cyclin C +/Δ (C-HET) and cyclin C Δ/Δ (C-KO) ES cells were probed with an antibody against phospho-Ser 2 and phospho-Ser 5 of RNA polymerase II CTD. Lower panel: immunoblotting with an anti-CTD antibody. ( c ) Cyclin C, CDK8, MED12 or MED13 were immunoprecipitated (IP) from wild-type or C-KO MEFs; the immunoblots were probed with the indicated antibodies, along with whole cell extracts (Input). ( d ) Scatterplot showing log of the normalized gene expression in control (x-axis) vs. C Δ/Δ (y-axis) MEFs, ES cells (ESC) and E18.5 brains. Parallel lines indicate 2-fold change (up- or down-regulation) in transcript levels. ( e ) Scatterplot showing log of the normalized gene expression values in control brains (x-axis) vs. control ES cells (y-axis). This is a control to panel d . It illustrates that different compartments (brain, ES cells) display very distinct gene expression patterns, as expected.

    Techniques Used: Expressing, Immunoprecipitation, In Vitro, Recombinant, Activity Assay, Autoradiography, Staining, Western Blot

    7) Product Images from "TRPM2 channel–mediated regulation of autophagy maintains mitochondrial function and promotes gastric cancer cell survival via the JNK-signaling pathway"

    Article Title: TRPM2 channel–mediated regulation of autophagy maintains mitochondrial function and promotes gastric cancer cell survival via the JNK-signaling pathway

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M117.817635

    Expression level of TRPM2 is negatively correlated with the overall survival rate of gastric cancer patients. The expression of TRPM2 was analyzed according to the Kaplan-Meier method using a median cutoff. Patients with TRPM2 mRNA levels higher than the median value were considered “high,” and patients with mRNA expression lower than the median were classified as “low.” Survival curves show the correlation between high TRPM2 expression and low patient survival. A, all patients; B, patients with stage I and II cancer and patients with stage III and IV gastric cancer. The hazard ratios generated are greater than 1 suggesting that patients with high TRPM2 expression will die at a higher rate in a given period of time than those with low TRPM2.
    Figure Legend Snippet: Expression level of TRPM2 is negatively correlated with the overall survival rate of gastric cancer patients. The expression of TRPM2 was analyzed according to the Kaplan-Meier method using a median cutoff. Patients with TRPM2 mRNA levels higher than the median value were considered “high,” and patients with mRNA expression lower than the median were classified as “low.” Survival curves show the correlation between high TRPM2 expression and low patient survival. A, all patients; B, patients with stage I and II cancer and patients with stage III and IV gastric cancer. The hazard ratios generated are greater than 1 suggesting that patients with high TRPM2 expression will die at a higher rate in a given period of time than those with low TRPM2.

    Techniques Used: Expressing, Generated

    Down-regulation of TRPM2 promotes cell death in AGS and MKN-45 cells. A , annexin V/7AAD staining of TRPM2 KD and Scr. cells 72 h after being seeded in 6-well plates. Dot plots represent the population of live cells ( lower left quadrant ), necrotic cells ( upper left quadrant ), apoptotic cells ( lower right quadrant ), and early necrotic or late apoptotic cells ( upper right quadrant ). B, bar graphs depict the quantification of apoptosis data ( n = 3). C and D, Western blot analysis of cleaved caspase-7 in TRPM2 KD cells. Statistical significance of Western blotting results was calculated as the relative ratio of cleaved caspase-7 protein normalized to β-actin ( n = 3). Asterisks indicated a significant difference from scrambler: ***, p
    Figure Legend Snippet: Down-regulation of TRPM2 promotes cell death in AGS and MKN-45 cells. A , annexin V/7AAD staining of TRPM2 KD and Scr. cells 72 h after being seeded in 6-well plates. Dot plots represent the population of live cells ( lower left quadrant ), necrotic cells ( upper left quadrant ), apoptotic cells ( lower right quadrant ), and early necrotic or late apoptotic cells ( upper right quadrant ). B, bar graphs depict the quantification of apoptosis data ( n = 3). C and D, Western blot analysis of cleaved caspase-7 in TRPM2 KD cells. Statistical significance of Western blotting results was calculated as the relative ratio of cleaved caspase-7 protein normalized to β-actin ( n = 3). Asterisks indicated a significant difference from scrambler: ***, p

    Techniques Used: Staining, Western Blot

    TRPM2 KD inhibits proliferation in AGS and MKN-45 cells. A and C, trypan blue counting of Scr. and TRPM2 knockdown cells at 24, 48, and 72 h after being seeded ( n = 4). B and D, MTT assay was used to quantify viable cells at 24, 48, and 72 h after being seeded ( n = 5). E and G, CFSE proliferation assay after 4 days of cell culturing. In the corresponding histograms, the x axis represents the CFSE fluorescent signal intensity, and the y axis shows the number of events. F and H, bar graphs representing CFSE mean of data from n = 3; ***, p
    Figure Legend Snippet: TRPM2 KD inhibits proliferation in AGS and MKN-45 cells. A and C, trypan blue counting of Scr. and TRPM2 knockdown cells at 24, 48, and 72 h after being seeded ( n = 4). B and D, MTT assay was used to quantify viable cells at 24, 48, and 72 h after being seeded ( n = 5). E and G, CFSE proliferation assay after 4 days of cell culturing. In the corresponding histograms, the x axis represents the CFSE fluorescent signal intensity, and the y axis shows the number of events. F and H, bar graphs representing CFSE mean of data from n = 3; ***, p

    Techniques Used: MTT Assay, Proliferation Assay, Cell Culture

    8) Product Images from "Abnormal Levels of Gadd45alpha in Developing Neocortex Impair Neurite Outgrowth"

    Article Title: Abnormal Levels of Gadd45alpha in Developing Neocortex Impair Neurite Outgrowth

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0044207

    Gadd45a expression is critical for development of normal neuron morphology in neocortex. E15.5 mouse cerebral cortex was electroporated with constructs designed to reduce or increase Gadd45a levels in electroporated cells. Each construct was co-electroporated with vectors encoding red (RFP) or green (GFP) fluorescent reporter protein. RFP or GFP-positive neurons were analyzed at P14 or P10 respectively (n = 4–6 brains/group from two separate litters). ( A and B ) Although most Gadd45a shRNA-treated neurons ( B ) reached the upper layers of neocortex, there were significantly more cells distributed beneath upper layers compared to RFP alone ( A ) (also see Fig. S8 ). Higher magnification of control neurons ( A’ and A” ) and Gadd45a shRNA-treated ( B’ and B” ) reveals that the dendritic processes of neurons expressing Gadd45a shRNA are less arborized than those of neurons expressing RFP alone. Arrows indicate somas of RFP positive neurons. ( C ) Migration of neurons to the upper layers (2/3) of neocortex is unaffected by overexpression of Gadd45a. Left panels show neurons in control brains. Right panels show neurons overexpressing Gadd45a. ( C’ ) Higher magnification images show examples of Gadd45a-AU1 neurons (right panels) with hypertrophied, multipolar shaped somas compared to control neurons (left panels). Scale bars = 50 µm ( A, A” ).
    Figure Legend Snippet: Gadd45a expression is critical for development of normal neuron morphology in neocortex. E15.5 mouse cerebral cortex was electroporated with constructs designed to reduce or increase Gadd45a levels in electroporated cells. Each construct was co-electroporated with vectors encoding red (RFP) or green (GFP) fluorescent reporter protein. RFP or GFP-positive neurons were analyzed at P14 or P10 respectively (n = 4–6 brains/group from two separate litters). ( A and B ) Although most Gadd45a shRNA-treated neurons ( B ) reached the upper layers of neocortex, there were significantly more cells distributed beneath upper layers compared to RFP alone ( A ) (also see Fig. S8 ). Higher magnification of control neurons ( A’ and A” ) and Gadd45a shRNA-treated ( B’ and B” ) reveals that the dendritic processes of neurons expressing Gadd45a shRNA are less arborized than those of neurons expressing RFP alone. Arrows indicate somas of RFP positive neurons. ( C ) Migration of neurons to the upper layers (2/3) of neocortex is unaffected by overexpression of Gadd45a. Left panels show neurons in control brains. Right panels show neurons overexpressing Gadd45a. ( C’ ) Higher magnification images show examples of Gadd45a-AU1 neurons (right panels) with hypertrophied, multipolar shaped somas compared to control neurons (left panels). Scale bars = 50 µm ( A, A” ).

    Techniques Used: Expressing, Construct, shRNA, Migration, Over Expression

    Gadd45a regulates neurite outgrowth from cortical neurons in vitro. ( A ) Method used to transfect, culture and analyze transfected cortical neurons. E15.5 mice were electroporated with cDNA encoding GFP and different Gadd45a constructs. At E16.5, GFP-positive regions of cortex were isolated, cultured 6DIV, fixed, imaged and analyzed. Sholl analyses were performed by centering concentric rings with increasing radii of 10 um over the soma center and counting the number of times the dendritic processes intersected the rings. ( B-D ) Images of GFP-positive cells converted for Sholl analyses. Examples of control (pLKO +GFP) ( B ), Gadd45a knockdown (Gadd45a shRNA + GFP) ( C ), and Gadd45a overexpressing (Gadd45a -AU1+GFP) ( D ) neurons. Scale bar = 20 µm. ( C and E ) Gadd45a knockdown results in significantly fewer neurites. ( D and F ) Gadd45a overexpression results in hypertrophied somas, and significantly fewer distal processes (∼70 µm –200 um from the soma). The significant difference within the first 10 µm is likely due to the increase in soma size (see Fig. S6 ). Note: Gadd45a overexpression (blue) was compared to two different controls, GFP (green) and pLKO (black, same data presented in (E)). ( E ) Electroporation of a Gadd45a-shRNA resistant construct (R- Gadd45a (blue line)) is able to significantly restore neuronal morphology compared to Gadd45a shRNA treated neurons. ( E and F ) Lines with asterisks indicate ranges where data was averaged for posthoc analysis (bar graphs). Sholl data was analyzed using two-way ANOVA with repeated measures while the averaged data was analyzed by Fisher’s PLSD post-hoc test. ns = not significant.
    Figure Legend Snippet: Gadd45a regulates neurite outgrowth from cortical neurons in vitro. ( A ) Method used to transfect, culture and analyze transfected cortical neurons. E15.5 mice were electroporated with cDNA encoding GFP and different Gadd45a constructs. At E16.5, GFP-positive regions of cortex were isolated, cultured 6DIV, fixed, imaged and analyzed. Sholl analyses were performed by centering concentric rings with increasing radii of 10 um over the soma center and counting the number of times the dendritic processes intersected the rings. ( B-D ) Images of GFP-positive cells converted for Sholl analyses. Examples of control (pLKO +GFP) ( B ), Gadd45a knockdown (Gadd45a shRNA + GFP) ( C ), and Gadd45a overexpressing (Gadd45a -AU1+GFP) ( D ) neurons. Scale bar = 20 µm. ( C and E ) Gadd45a knockdown results in significantly fewer neurites. ( D and F ) Gadd45a overexpression results in hypertrophied somas, and significantly fewer distal processes (∼70 µm –200 um from the soma). The significant difference within the first 10 µm is likely due to the increase in soma size (see Fig. S6 ). Note: Gadd45a overexpression (blue) was compared to two different controls, GFP (green) and pLKO (black, same data presented in (E)). ( E ) Electroporation of a Gadd45a-shRNA resistant construct (R- Gadd45a (blue line)) is able to significantly restore neuronal morphology compared to Gadd45a shRNA treated neurons. ( E and F ) Lines with asterisks indicate ranges where data was averaged for posthoc analysis (bar graphs). Sholl data was analyzed using two-way ANOVA with repeated measures while the averaged data was analyzed by Fisher’s PLSD post-hoc test. ns = not significant.

    Techniques Used: In Vitro, Transfection, Mouse Assay, Construct, Isolation, Cell Culture, shRNA, Over Expression, Electroporation

    9) Product Images from "Centrobin-mediated Regulation of the Centrosomal Protein 4.1-associated Protein (CPAP) Level Limits Centriole Length during Elongation Stage *"

    Article Title: Centrobin-mediated Regulation of the Centrosomal Protein 4.1-associated Protein (CPAP) Level Limits Centriole Length during Elongation Stage *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M114.603423

    Centrobin overexpression results in increased cellular CPAP but not CP110 and hSAS-6 levels. A , 293T cells transfected with control or myc-centrobin expression vectors were lysed after 72 h of transfection and immunoblotted ( IB ) using the anti-CPAP, -CP110,
    Figure Legend Snippet: Centrobin overexpression results in increased cellular CPAP but not CP110 and hSAS-6 levels. A , 293T cells transfected with control or myc-centrobin expression vectors were lysed after 72 h of transfection and immunoblotted ( IB ) using the anti-CPAP, -CP110,

    Techniques Used: Over Expression, Transfection, Expressing

    10) Product Images from "The Groucho-associated Phosphatase PPM1B Displaces Pax Transactivation Domain Interacting Protein (PTIP) to Switch the Transcription Factor Pax2 from a Transcriptional Activator to a Repressor *"

    Article Title: The Groucho-associated Phosphatase PPM1B Displaces Pax Transactivation Domain Interacting Protein (PTIP) to Switch the Transcription Factor Pax2 from a Transcriptional Activator to a Repressor *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M114.607424

    Grg4 and PPM1B co-IP. Rabbit anti-PPM1B or goat anti-Grg4 were used to IP proteins from nuclear extracts directly in the presence of EtBr and Western blotted ( WB ) as indicated. Controls are rabbit IgG for PPM1B and goat IgG for Grg4.
    Figure Legend Snippet: Grg4 and PPM1B co-IP. Rabbit anti-PPM1B or goat anti-Grg4 were used to IP proteins from nuclear extracts directly in the presence of EtBr and Western blotted ( WB ) as indicated. Controls are rabbit IgG for PPM1B and goat IgG for Grg4.

    Techniques Used: Co-Immunoprecipitation Assay, Western Blot

    PPM1B is required for Pax2- and Grg4-mediated gene repression. A , control negative sh GFP reporter cells or GFP reporter cells with shRNA 1, 2, or 4 were transfected with Grg4, Pax2, wild-type, or phosphatase-deficient PPM1B as indicated. Whole cell lysates were Western blotted ( WB ) for the indicated proteins with β-actin used as a loading control. Note that GFP expression increased upon Pax2 transfection ( lanes 2 , 7 , 12 , and 17 ) in all cell lines; this activation was inhibited by Grg4 ( lane 3 ) in control cells but not in PPM1B knockdown cells ( lanes 8 , 13 , and 18 ). The Grg4-mediated inhibition was rescued with wild-type PPM1B in the knockdown cells ( lanes 9 , 14 , and 19 ) but not mutant enzyme ( lanes 10 , 15 , and 20 ). B , scanning densitometry for GFP expression quantitated by ImageJ is shown. GFP expression in the CMV empty vector alone ( lanes 1 , 6 , 11 , and 16 ) are normalized to 1, and expression in other lysates is relative to the respective control. Note that GFP expression is completely suppressed in the control sh cells transfected with Pax2/Grg4 in the presence or absence of PPM1B or mutant PPM1B (compare lane 2 with lanes 3–5 ). In the knock-out cells, co-expression of Pax2/Grg4 did not repress GFP expression (compare lanes 7 and 8 ; lanes 12 and 13 ; and lanes 17 and 18 ). Wild-type ( lanes 9 , 14 , and 19 ) but not mutant PPM1B ( lanes 10 , 15 , and 20 ) restores repression of GFP. All bars are averages of duplicates with error bars indicating S.D. Note comparisons are made between all groups of transfections in each cell line, and significant comparisons are shown. *, p
    Figure Legend Snippet: PPM1B is required for Pax2- and Grg4-mediated gene repression. A , control negative sh GFP reporter cells or GFP reporter cells with shRNA 1, 2, or 4 were transfected with Grg4, Pax2, wild-type, or phosphatase-deficient PPM1B as indicated. Whole cell lysates were Western blotted ( WB ) for the indicated proteins with β-actin used as a loading control. Note that GFP expression increased upon Pax2 transfection ( lanes 2 , 7 , 12 , and 17 ) in all cell lines; this activation was inhibited by Grg4 ( lane 3 ) in control cells but not in PPM1B knockdown cells ( lanes 8 , 13 , and 18 ). The Grg4-mediated inhibition was rescued with wild-type PPM1B in the knockdown cells ( lanes 9 , 14 , and 19 ) but not mutant enzyme ( lanes 10 , 15 , and 20 ). B , scanning densitometry for GFP expression quantitated by ImageJ is shown. GFP expression in the CMV empty vector alone ( lanes 1 , 6 , 11 , and 16 ) are normalized to 1, and expression in other lysates is relative to the respective control. Note that GFP expression is completely suppressed in the control sh cells transfected with Pax2/Grg4 in the presence or absence of PPM1B or mutant PPM1B (compare lane 2 with lanes 3–5 ). In the knock-out cells, co-expression of Pax2/Grg4 did not repress GFP expression (compare lanes 7 and 8 ; lanes 12 and 13 ; and lanes 17 and 18 ). Wild-type ( lanes 9 , 14 , and 19 ) but not mutant PPM1B ( lanes 10 , 15 , and 20 ) restores repression of GFP. All bars are averages of duplicates with error bars indicating S.D. Note comparisons are made between all groups of transfections in each cell line, and significant comparisons are shown. *, p

    Techniques Used: shRNA, Transfection, Western Blot, Expressing, Activation Assay, Inhibition, Mutagenesis, Plasmid Preparation, Knock-Out

    Pax2/Grg4-mediated chromatin remodeling at the Rap1A locus is PPM1B-dependent. A , GFP reporter cells with the control sh or cells with PPM1B shRNA 1 and shRNA 4 were transfected as noted and Western blotted ( WB ) for Grg4, PPM1B, and Pax2. Loading was normalized for β-galactosidase activity. B , RAP1A mRNA expression in the transfected cells from A . Rap1A expression in the CMV empty vector alone ( lanes 1 , 6 , and 11 ) are normalized to 1 and expression in other lanes are relative to the respective control. Note that in all cell lines, Pax2 stimulates Rap1a expression ( lanes 2 , 7 , and 12 ). In control cells, Pax2-Grg4 co-expression represses Rap1A expression ( lane 3 ) but not in the absence of PPM1B ( lanes 8 ,13). Rescue with wild-type PPM1B ( lanes 9 and 14 ) but not phosphatase-deficient PPM1B ( lanes 10 and 15 ) restores repression of Rap1A expression in the knock-out cells. All bars are averages of two experiments with PCR done in triplicate and error bars indicating S.D. Note comparisons are made between all groups of transfections in each cell line, and significant comparisons are shown. *, p
    Figure Legend Snippet: Pax2/Grg4-mediated chromatin remodeling at the Rap1A locus is PPM1B-dependent. A , GFP reporter cells with the control sh or cells with PPM1B shRNA 1 and shRNA 4 were transfected as noted and Western blotted ( WB ) for Grg4, PPM1B, and Pax2. Loading was normalized for β-galactosidase activity. B , RAP1A mRNA expression in the transfected cells from A . Rap1A expression in the CMV empty vector alone ( lanes 1 , 6 , and 11 ) are normalized to 1 and expression in other lanes are relative to the respective control. Note that in all cell lines, Pax2 stimulates Rap1a expression ( lanes 2 , 7 , and 12 ). In control cells, Pax2-Grg4 co-expression represses Rap1A expression ( lane 3 ) but not in the absence of PPM1B ( lanes 8 ,13). Rescue with wild-type PPM1B ( lanes 9 and 14 ) but not phosphatase-deficient PPM1B ( lanes 10 and 15 ) restores repression of Rap1A expression in the knock-out cells. All bars are averages of two experiments with PCR done in triplicate and error bars indicating S.D. Note comparisons are made between all groups of transfections in each cell line, and significant comparisons are shown. *, p

    Techniques Used: shRNA, Transfection, Western Blot, Activity Assay, Expressing, Plasmid Preparation, Knock-Out, Polymerase Chain Reaction

    11) Product Images from "The PRMT5/WDR77 complex regulates alternative splicing through ZNF326 in breast cancer"

    Article Title: The PRMT5/WDR77 complex regulates alternative splicing through ZNF326 in breast cancer

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx727

    Expression of PRMT5 and WDR77 in breast cancer ( A ). Expression analysis of 109 paired samples from The Cancer Genome Atlas (TCGA) database shows significant overexpression of PRMT5 and WDR77 in breast cancer samples relative to matched normal samples ( B ). Fold expression of WDR77 and PRMT5 in breast normal (MCF10A), ER+ (MCF7, T47D) and ER- (MDA-MB-231, HCC38) breast cancer cell lines. Normalized to GAPDH ( C ). Immunoblotting of WDR77 and PRMT5 in nuclear and cytoplasmic extracts of breast normal and cancer cell lines. Actin was used as loading control. ( D ). Fold expression (relative to GAPDH) of WDR77 and PRMT5 and ( E ). Immunoblotting of the respective proteins in sh Scrambled, sh WDR77 and sh PRMT5-treated samples. Actin was used as loading control in E ( F ). (Top panel) Annexin V staining showing increased number of apoptotic cells upon loss of WDR77 and PRMT5 . (Bottom panel) Box plots of results from apoptosis assay for three biological replicates. Student's t-test * P
    Figure Legend Snippet: Expression of PRMT5 and WDR77 in breast cancer ( A ). Expression analysis of 109 paired samples from The Cancer Genome Atlas (TCGA) database shows significant overexpression of PRMT5 and WDR77 in breast cancer samples relative to matched normal samples ( B ). Fold expression of WDR77 and PRMT5 in breast normal (MCF10A), ER+ (MCF7, T47D) and ER- (MDA-MB-231, HCC38) breast cancer cell lines. Normalized to GAPDH ( C ). Immunoblotting of WDR77 and PRMT5 in nuclear and cytoplasmic extracts of breast normal and cancer cell lines. Actin was used as loading control. ( D ). Fold expression (relative to GAPDH) of WDR77 and PRMT5 and ( E ). Immunoblotting of the respective proteins in sh Scrambled, sh WDR77 and sh PRMT5-treated samples. Actin was used as loading control in E ( F ). (Top panel) Annexin V staining showing increased number of apoptotic cells upon loss of WDR77 and PRMT5 . (Bottom panel) Box plots of results from apoptosis assay for three biological replicates. Student's t-test * P

    Techniques Used: Expressing, Over Expression, Multiple Displacement Amplification, Staining, Apoptosis Assay

    Loss of PRMT5 and WDR77 leads to defects in alternative splicing and inclusion of A-T rich exons ( A and B ). (Top) Frequency of A or T upstream and downstream from splice sites of included exons (blue) excluded exons (green) and unaffected control exons (red). The dotted black line marks the meeting point of upstream and downstream datasets. (Below) Frequency of 5-base oligonucleotides in the regions around splice sites of included ( x -axis) versus control ( y -axis) exons in sh WDR77 (Left) and sh PRMT5 (Right) samples. Scatter plot of genes that are alternatively spliced and up- or downregulated upon loss of WDR77 ( C ) and PRMT5 ( D ) relative to scrambled shRNA control (Red- > 1.5-fold upregulated Blue- > 1.5-fold downregulated).
    Figure Legend Snippet: Loss of PRMT5 and WDR77 leads to defects in alternative splicing and inclusion of A-T rich exons ( A and B ). (Top) Frequency of A or T upstream and downstream from splice sites of included exons (blue) excluded exons (green) and unaffected control exons (red). The dotted black line marks the meeting point of upstream and downstream datasets. (Below) Frequency of 5-base oligonucleotides in the regions around splice sites of included ( x -axis) versus control ( y -axis) exons in sh WDR77 (Left) and sh PRMT5 (Right) samples. Scatter plot of genes that are alternatively spliced and up- or downregulated upon loss of WDR77 ( C ) and PRMT5 ( D ) relative to scrambled shRNA control (Red- > 1.5-fold upregulated Blue- > 1.5-fold downregulated).

    Techniques Used: shRNA

    Alternative splicing coupled mRNA decay of transcripts upon loss of PRMT5 . qPCR analysis showing (from left to right) relative levels of exon inclusion, pre-mRNA and the mRNA/pre-mRNA ratio in sh Scrambled and sh PRMT5 samples for ( A ) REPIN1/AP4 ( B ) ST3GAL6 ( C ) PFKM ( D ) TRNAU1AP/SECP43 . The illustrations depict the gene structure with exons shown as black boxes and introns as lines. The included exons are shown in red. Arrows indicate regions to which primers were designed. Student's t -test * P
    Figure Legend Snippet: Alternative splicing coupled mRNA decay of transcripts upon loss of PRMT5 . qPCR analysis showing (from left to right) relative levels of exon inclusion, pre-mRNA and the mRNA/pre-mRNA ratio in sh Scrambled and sh PRMT5 samples for ( A ) REPIN1/AP4 ( B ) ST3GAL6 ( C ) PFKM ( D ) TRNAU1AP/SECP43 . The illustrations depict the gene structure with exons shown as black boxes and introns as lines. The included exons are shown in red. Arrows indicate regions to which primers were designed. Student's t -test * P

    Techniques Used: Real-time Polymerase Chain Reaction

    ZNF326 is symmetrically dimethylated at R175 by the PRMT5/WDR77 complex ( A ). ZNF326 has two glycine-arginine rich motifs. Asterisk indicates R175 that was identified to be dimethylated ( B ). Immunoblots of immunoprecipitates showing symmetric dimethylation of ZNF326 ( C ). Representative tandem mass spectrum of the peptide GR(28.0314)GTPAYPESTFGSR {m/z:537.602 (+3). The fragment ion matching within 10 ppm are shown as either the B-ion (purple) or Y-ion (blue) series. The green dashed line indicates precursor m/z. The dotted lines in the fragmentation ladder sequence on the top the spectrum corresponds to the missing B-ion (purple) and Y-ion (blue) series ( D ). Mass Spectrometry analysis showing the relative abundance of random and dimethylated peptides in cells infected with sh Scrambled, sh WDR77 and sh PRMT5 and ( E ). Graphical representation of the same.
    Figure Legend Snippet: ZNF326 is symmetrically dimethylated at R175 by the PRMT5/WDR77 complex ( A ). ZNF326 has two glycine-arginine rich motifs. Asterisk indicates R175 that was identified to be dimethylated ( B ). Immunoblots of immunoprecipitates showing symmetric dimethylation of ZNF326 ( C ). Representative tandem mass spectrum of the peptide GR(28.0314)GTPAYPESTFGSR {m/z:537.602 (+3). The fragment ion matching within 10 ppm are shown as either the B-ion (purple) or Y-ion (blue) series. The green dashed line indicates precursor m/z. The dotted lines in the fragmentation ladder sequence on the top the spectrum corresponds to the missing B-ion (purple) and Y-ion (blue) series ( D ). Mass Spectrometry analysis showing the relative abundance of random and dimethylated peptides in cells infected with sh Scrambled, sh WDR77 and sh PRMT5 and ( E ). Graphical representation of the same.

    Techniques Used: Western Blot, Sequencing, Mass Spectrometry, Infection

    Networks of top interaction partners of WDR77 in the cytoplasm and nucleus identified by LC-MS/MS ( A ). Gene ontology analysis ( B ). Protein interaction network of top 15 interacting partners of WDR77 in the cytoplasm (red lines: newly identified interactions, black lines: previously characterized interactions) ( C ). Gene ontology analysis ( D ). Protein interaction network of top 15 interacting partners of WDR77 in the nucleus (red lines: newly identified interactions, black lines: previously characterized interactions) ( E ). Immunoblots of co-immunoprecipitations of WDR77, PRMT5 and ZNF326 (IP-Immunoprecipitation, WB-western blot).
    Figure Legend Snippet: Networks of top interaction partners of WDR77 in the cytoplasm and nucleus identified by LC-MS/MS ( A ). Gene ontology analysis ( B ). Protein interaction network of top 15 interacting partners of WDR77 in the cytoplasm (red lines: newly identified interactions, black lines: previously characterized interactions) ( C ). Gene ontology analysis ( D ). Protein interaction network of top 15 interacting partners of WDR77 in the nucleus (red lines: newly identified interactions, black lines: previously characterized interactions) ( E ). Immunoblots of co-immunoprecipitations of WDR77, PRMT5 and ZNF326 (IP-Immunoprecipitation, WB-western blot).

    Techniques Used: Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Western Blot, Immunoprecipitation

    The PRMT5/WDR77 complex shapes the transcriptome of MDA-MB-231 cells through methylation of ZNF326. Methylation of ZNF326 by the PRMT5/WDR77 complex is essential for Pol II transcription across A-T rich genes. Loss of PRMT5 or WDR77 leads to a loss of methylation of ZNF326 that results in slow progression of Pol II causing the inclusion of A-T rich exons in target genes. A subset of these transcripts is targeted for degradation thereby altering the shape of the transcriptome of the cell. ( A ) Represents the influence of PRMT5/WDR77 to coordinate the rate at which transcription may help determine splicing patterns, where, the absence of PRMT5/WDR77 ( B ) effects the rate and aberrant inclusion of exons.
    Figure Legend Snippet: The PRMT5/WDR77 complex shapes the transcriptome of MDA-MB-231 cells through methylation of ZNF326. Methylation of ZNF326 by the PRMT5/WDR77 complex is essential for Pol II transcription across A-T rich genes. Loss of PRMT5 or WDR77 leads to a loss of methylation of ZNF326 that results in slow progression of Pol II causing the inclusion of A-T rich exons in target genes. A subset of these transcripts is targeted for degradation thereby altering the shape of the transcriptome of the cell. ( A ) Represents the influence of PRMT5/WDR77 to coordinate the rate at which transcription may help determine splicing patterns, where, the absence of PRMT5/WDR77 ( B ) effects the rate and aberrant inclusion of exons.

    Techniques Used: Multiple Displacement Amplification, Methylation

    12) Product Images from "TNFSF14/LIGHT, a Non-Canonical NF-κB Stimulus, Induces the HIF Pathway"

    Article Title: TNFSF14/LIGHT, a Non-Canonical NF-κB Stimulus, Induces the HIF Pathway

    Journal: Cells

    doi: 10.3390/cells7080102

    LIGHT, a non-canonical NF-κB inducer, induces HIF expression and activity. ( A ) HeLa cells were treated with 100 ng/mL LIGHT for 0, 4 and 24 h. Whole cell lysates were prepared and analysed by western blot for the indicated non-canonical NF-κB pathway regulators, subunits and target genes. β-Actin was used as loading control. ( B ) HeLa cells were treated with 100 ng/mL LIGHT for 0, 4 and 24 h prior mRNA extraction and RT-qPCR analysis for RANTES transcript, normalised to Actin mRNA levels. All the values were normalised to the untreated sample. The graphs depict mean and SEM determined from at least three independent experiments. One-way Anova analysis was performed and significance determined as follows: ns = not significant, *** p ≤ 0.001. ( C ) HeLa cells, stably transfected with HRE luciferase reporter, were treated with 100 ng/mL LIGHT for 8 and 24 h prior to luciferase measurements. All of the values were normalised to the untreated sample. Graph depicts mean and SEM of a minimum of three independent biological experiments. One way Anova analysis was performed and significance determined as follows: ns = not significant, *** p ≤ 0.001. ( D ) HeLa and A549 cells were treated with 100 ng/mL LIGHT for 0, 4 and 24 h prior collection of whole cell lysates and western blot analysis for the depicted proteins. β-Actin was used as loading control. ( E ) HeLa cells were treated with 10 ng/mL TNF-α and 100 ng/mL LIGHT for 0, 4 and 24 h. Then, whole cell lysates were collected and western blot analysis was performed for the depicted proteins. β-Actin was used as loading control. ( F ) HeLa and A549 cells were treated with 100 ng/mL LIGHT for 0, 4 and 24 h. Then, whole cell lysates were collected and western blot analysis was performed for a subset of HIF-α specific targets. β-Actin was used as loading control. ( G ) HeLa cells and ( H ) A549 cells were treated with 100 ng/mL LIGHT for 0, 4 and 24 h, prior mRNA extraction and RT-qPCR analysis for PHD2 gene transcript, normalised to Actin mRNA levels. All the values were normalised to the untreated samples. The graph depicts mean and SEM determined from at least three independent biological experiments. One-way Anova analysis was performed and significance determined as follows: ns = not significant, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. ( I ) Analysis of canonical NF-κB signalling following NIK expression vectors for 48 h prior to luciferase measurements. All the values were normalised to the control sample. Graph depicts mean and SEM of a minimum of three independent biological experiments. Student t -test analysis was performed and significance determined as follows: ns = not significant, ** p ≤ 0.01. ( J ) HeLa cells were transfected with control and NIK expression vectors for 48 h prior to cell lysis. Western blot analysis was performed using the indicating antibodies. β-Actin was used as loading control.
    Figure Legend Snippet: LIGHT, a non-canonical NF-κB inducer, induces HIF expression and activity. ( A ) HeLa cells were treated with 100 ng/mL LIGHT for 0, 4 and 24 h. Whole cell lysates were prepared and analysed by western blot for the indicated non-canonical NF-κB pathway regulators, subunits and target genes. β-Actin was used as loading control. ( B ) HeLa cells were treated with 100 ng/mL LIGHT for 0, 4 and 24 h prior mRNA extraction and RT-qPCR analysis for RANTES transcript, normalised to Actin mRNA levels. All the values were normalised to the untreated sample. The graphs depict mean and SEM determined from at least three independent experiments. One-way Anova analysis was performed and significance determined as follows: ns = not significant, *** p ≤ 0.001. ( C ) HeLa cells, stably transfected with HRE luciferase reporter, were treated with 100 ng/mL LIGHT for 8 and 24 h prior to luciferase measurements. All of the values were normalised to the untreated sample. Graph depicts mean and SEM of a minimum of three independent biological experiments. One way Anova analysis was performed and significance determined as follows: ns = not significant, *** p ≤ 0.001. ( D ) HeLa and A549 cells were treated with 100 ng/mL LIGHT for 0, 4 and 24 h prior collection of whole cell lysates and western blot analysis for the depicted proteins. β-Actin was used as loading control. ( E ) HeLa cells were treated with 10 ng/mL TNF-α and 100 ng/mL LIGHT for 0, 4 and 24 h. Then, whole cell lysates were collected and western blot analysis was performed for the depicted proteins. β-Actin was used as loading control. ( F ) HeLa and A549 cells were treated with 100 ng/mL LIGHT for 0, 4 and 24 h. Then, whole cell lysates were collected and western blot analysis was performed for a subset of HIF-α specific targets. β-Actin was used as loading control. ( G ) HeLa cells and ( H ) A549 cells were treated with 100 ng/mL LIGHT for 0, 4 and 24 h, prior mRNA extraction and RT-qPCR analysis for PHD2 gene transcript, normalised to Actin mRNA levels. All the values were normalised to the untreated samples. The graph depicts mean and SEM determined from at least three independent biological experiments. One-way Anova analysis was performed and significance determined as follows: ns = not significant, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. ( I ) Analysis of canonical NF-κB signalling following NIK expression vectors for 48 h prior to luciferase measurements. All the values were normalised to the control sample. Graph depicts mean and SEM of a minimum of three independent biological experiments. Student t -test analysis was performed and significance determined as follows: ns = not significant, ** p ≤ 0.01. ( J ) HeLa cells were transfected with control and NIK expression vectors for 48 h prior to cell lysis. Western blot analysis was performed using the indicating antibodies. β-Actin was used as loading control.

    Techniques Used: Expressing, Activity Assay, Western Blot, Quantitative RT-PCR, Stable Transfection, Transfection, Luciferase, Lysis

    13) Product Images from "The ATR-mediated S phase checkpoint prevents rereplication in mammalian cells when licensing control is disrupted"

    Article Title: The ATR-mediated S phase checkpoint prevents rereplication in mammalian cells when licensing control is disrupted

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200704138

    RPA2, MCM2, and Rb are important effector proteins downstream of ATR to inhibit Cdt1-induced rereplication. (A) 48 h after Ad-vec or Ad-Cdt1 infection, chromatin was purified from U2OS cells expressing shRNA-ATR, shRNA-ATM, or vector MKO (left), or cell lysates were prepared from U2OS or IMR90 cells (right). The adenovirus titers U2OS (5 × 10 7 pfu/ml, MOI = 25) and IMR90 (6 × 10 8 pfu/ml, MOI = 600) were used. After 1 h, 50 J/m 2 UV treatment was used as a positive control for MCM2 phosphorylation. Western blot analyses were performed as indicated. RPA2p, phosphorylated RPA2; MCM2-pS108, phosphorylated MCM2 at S108. (B) U2OS cell lines expressing the HA-RPA2 wild type or HA-RPA2-phospho mutant (S4A/S8A/S11A/S12A/S13A/T21A/S33A) with endogenous RPA2 silenced by shRNA were generated as described previously ( Olson et al., 2006 ). FACS analyses were performed 48 h after Ad-vec or Ad-Cdt1 infection (5 × 10 7 pfu/ml, MOI = 25). Western blot analyses were performed as indicated. endo-RPA2, endogenous RPA2; HA-RPA2p, phosphorylated HA-tagged RPA2. (C) U2OS cells carrying tetracycline-regulated constitutively active Rb were infected with Ad-vec or Ad-Cdt1 (5 × 10 7 pfu/ml, MOI = 25) in the presence of tetracycline (+tet, un-Rb off) or 24 h after removal of tetracycline (−tet, un-Rb on). 36 h after infection, cell cycle profiles were monitored by FACS analysis (top). Rb was silenced in A549 and T98G cells by expressing shRNA from retroviral vector MKO. FACS analysis was performed 48 h after Ad-vec or Ad-Cdt1 infection (bottom; A549: 6 × 10 8 pfu/ml, MOI = 400; T98G: 6 × 10 8 pfu/ml, MOI = 460). (D) Cell lysates were prepared from IMR90 or A549 cells expressing ATR-shRNA or vector MKO 20 or 40 h after Ad-vec or Ad-Cdt1 infection (6 × 10 8 pfu/ml; IMR90, MOI = 600; A549, MOI = 400). Rb phosphorylation was analyzed by the antibody G99-549, which specifically recognizes nonphosphorylated S608 species. Ku80 was used as a loading control.
    Figure Legend Snippet: RPA2, MCM2, and Rb are important effector proteins downstream of ATR to inhibit Cdt1-induced rereplication. (A) 48 h after Ad-vec or Ad-Cdt1 infection, chromatin was purified from U2OS cells expressing shRNA-ATR, shRNA-ATM, or vector MKO (left), or cell lysates were prepared from U2OS or IMR90 cells (right). The adenovirus titers U2OS (5 × 10 7 pfu/ml, MOI = 25) and IMR90 (6 × 10 8 pfu/ml, MOI = 600) were used. After 1 h, 50 J/m 2 UV treatment was used as a positive control for MCM2 phosphorylation. Western blot analyses were performed as indicated. RPA2p, phosphorylated RPA2; MCM2-pS108, phosphorylated MCM2 at S108. (B) U2OS cell lines expressing the HA-RPA2 wild type or HA-RPA2-phospho mutant (S4A/S8A/S11A/S12A/S13A/T21A/S33A) with endogenous RPA2 silenced by shRNA were generated as described previously ( Olson et al., 2006 ). FACS analyses were performed 48 h after Ad-vec or Ad-Cdt1 infection (5 × 10 7 pfu/ml, MOI = 25). Western blot analyses were performed as indicated. endo-RPA2, endogenous RPA2; HA-RPA2p, phosphorylated HA-tagged RPA2. (C) U2OS cells carrying tetracycline-regulated constitutively active Rb were infected with Ad-vec or Ad-Cdt1 (5 × 10 7 pfu/ml, MOI = 25) in the presence of tetracycline (+tet, un-Rb off) or 24 h after removal of tetracycline (−tet, un-Rb on). 36 h after infection, cell cycle profiles were monitored by FACS analysis (top). Rb was silenced in A549 and T98G cells by expressing shRNA from retroviral vector MKO. FACS analysis was performed 48 h after Ad-vec or Ad-Cdt1 infection (bottom; A549: 6 × 10 8 pfu/ml, MOI = 400; T98G: 6 × 10 8 pfu/ml, MOI = 460). (D) Cell lysates were prepared from IMR90 or A549 cells expressing ATR-shRNA or vector MKO 20 or 40 h after Ad-vec or Ad-Cdt1 infection (6 × 10 8 pfu/ml; IMR90, MOI = 600; A549, MOI = 400). Rb phosphorylation was analyzed by the antibody G99-549, which specifically recognizes nonphosphorylated S608 species. Ku80 was used as a loading control.

    Techniques Used: Infection, Purification, Expressing, shRNA, Plasmid Preparation, Positive Control, Western Blot, Mutagenesis, Generated, FACS

    14) Product Images from "The arginine methyltransferase CARM1 represses p300•ACT•CREMτ activity and is required for spermiogenesis"

    Article Title: The arginine methyltransferase CARM1 represses p300•ACT•CREMτ activity and is required for spermiogenesis

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky240

    Carm1 expression and localization in testis. ( A ) Carm1 mRNA expression levels in different human tissues as detected by RNA-seq from GTEx database; RPKM, Reads Per Kilobase of transcript per Million; ( B ) Relative mRNA levels of Carm1 quantified by quantitative real time PCR (qPCR). Testicular samples were collected at embryonic day 12 (E12) and E15, as well as postnatal day 0 (P0), P7, P14, P21 and P42. The mRNA levels at E12 were arbitrarily set as 1; Data were calculated from biological triplicates and shown as mean±SD. ( C ) Relative mRNA levels of Carm1 quantified by qPCR in purified germ cell populations from the testis; The mRNA levels in Sertoli cells were arbitrarily set as 1. Data were calculated from three biological replicates with 5 mice in each pool, and shown as mean ± SD. ( D ) Expression and localization of Carm1 protein by immunofluorescent staining (IF) using Carm1-specific antibody. Carm1 protein localization in three representative stages of spermatogenic cycles is presented in seminiferous tubules. The expression intensity and localization of CARM1 were summarized in ( E ). Bar, 10 μm.
    Figure Legend Snippet: Carm1 expression and localization in testis. ( A ) Carm1 mRNA expression levels in different human tissues as detected by RNA-seq from GTEx database; RPKM, Reads Per Kilobase of transcript per Million; ( B ) Relative mRNA levels of Carm1 quantified by quantitative real time PCR (qPCR). Testicular samples were collected at embryonic day 12 (E12) and E15, as well as postnatal day 0 (P0), P7, P14, P21 and P42. The mRNA levels at E12 were arbitrarily set as 1; Data were calculated from biological triplicates and shown as mean±SD. ( C ) Relative mRNA levels of Carm1 quantified by qPCR in purified germ cell populations from the testis; The mRNA levels in Sertoli cells were arbitrarily set as 1. Data were calculated from three biological replicates with 5 mice in each pool, and shown as mean ± SD. ( D ) Expression and localization of Carm1 protein by immunofluorescent staining (IF) using Carm1-specific antibody. Carm1 protein localization in three representative stages of spermatogenic cycles is presented in seminiferous tubules. The expression intensity and localization of CARM1 were summarized in ( E ). Bar, 10 μm.

    Techniques Used: Expressing, RNA Sequencing Assay, Real-time Polymerase Chain Reaction, Purification, Mouse Assay, Staining

    Genome-wide changes of the mRNA transcriptome in the purified round spermatids [RS] and elongating/condensing spermatids [ECS] upon Carm1 cKO. ( A ) A snapshot of the UCSC genome browser for the Carm1 mRNA expression track in the round spermatids between WT and cKO. Gray rectangle highlights the two floxed exons that were specifically deleted in the cKO haploid spermatids. ( B ) Scatter plots showing the differentially expressed genes (DEGs) (Cutoff: fold change ≥ 2, FDR
    Figure Legend Snippet: Genome-wide changes of the mRNA transcriptome in the purified round spermatids [RS] and elongating/condensing spermatids [ECS] upon Carm1 cKO. ( A ) A snapshot of the UCSC genome browser for the Carm1 mRNA expression track in the round spermatids between WT and cKO. Gray rectangle highlights the two floxed exons that were specifically deleted in the cKO haploid spermatids. ( B ) Scatter plots showing the differentially expressed genes (DEGs) (Cutoff: fold change ≥ 2, FDR

    Techniques Used: Genome Wide, Purification, Expressing

    Luciferase reporter assay showing that CARM1 represses transcriptional activity of testis-specific CREMτ, ACT and CBP/p300. ( A ) Schematic diagram showing the structure of CREMτ-responsive luciferase reporter CRE/NR-PRL-pGL. The consensus sequence (cAMP-responsive element, CRE, and nuclear receptor, NR) for the DNA binding of CREMτ and GCNF transcription factors was inserted upstream of a minimal rat Prolactin promoter (PRL) in the pGL3.0 luciferase vector (Promega). ( B–E ) Relative fold changes of luciferase expression calculated from the transfection with different combinations of plasmids as indicated. Plasmids were generated as described in methods and materials. Dual-luciferase activity of three independent biological replicates were recorded for each assay. Renilla luciferase activity was used as the internal control. Data were presented as mean ± SEM. * P
    Figure Legend Snippet: Luciferase reporter assay showing that CARM1 represses transcriptional activity of testis-specific CREMτ, ACT and CBP/p300. ( A ) Schematic diagram showing the structure of CREMτ-responsive luciferase reporter CRE/NR-PRL-pGL. The consensus sequence (cAMP-responsive element, CRE, and nuclear receptor, NR) for the DNA binding of CREMτ and GCNF transcription factors was inserted upstream of a minimal rat Prolactin promoter (PRL) in the pGL3.0 luciferase vector (Promega). ( B–E ) Relative fold changes of luciferase expression calculated from the transfection with different combinations of plasmids as indicated. Plasmids were generated as described in methods and materials. Dual-luciferase activity of three independent biological replicates were recorded for each assay. Renilla luciferase activity was used as the internal control. Data were presented as mean ± SEM. * P

    Techniques Used: Luciferase, Reporter Assay, Activity Assay, Activated Clotting Time Assay, Sequencing, Binding Assay, Plasmid Preparation, Expressing, Transfection, Generated

    Defective development of haploid spermatids in Carm1 cKO mice. ( A ) Periodic acid–Schiff (PAS) staining of the cross-sections of paraffin-embedded testes from WT and cKO mice. Z, Zygotene; P, Pachytene; RS, Round spermatids; ES, Elongating spermatids. ( B ) Statistic comparison of the average number of spermatids relative to Sertoli cells, calculated from PAS-stained cross-sections. Data were presented as mean ± SEM. * P
    Figure Legend Snippet: Defective development of haploid spermatids in Carm1 cKO mice. ( A ) Periodic acid–Schiff (PAS) staining of the cross-sections of paraffin-embedded testes from WT and cKO mice. Z, Zygotene; P, Pachytene; RS, Round spermatids; ES, Elongating spermatids. ( B ) Statistic comparison of the average number of spermatids relative to Sertoli cells, calculated from PAS-stained cross-sections. Data were presented as mean ± SEM. * P

    Techniques Used: Mouse Assay, Staining

    Phenotypic analysis of germline-specific conditional Carm1 KO (cKO) mouse model. ( A ) Gross morphology of the testis and epididymis from WT and cKO mice. ( B ) Comparison of the average sperm count in the cauda epididymis between the WT and cKO mice at 6-week-old age. Three biological repeats were performed. Data were shown as mean ± SD. ( C ) Representative Hematoxylin and Eosin (H E) staining of cauda epididymis and ( D ) phase-contrast microscopy of cauda sperm from WT and cKO littermates. Note that the majority of sperm exhibit deformed head morphology in the cKO cauda epididymis, as indicated. Data were presented as mean ± SEM.
    Figure Legend Snippet: Phenotypic analysis of germline-specific conditional Carm1 KO (cKO) mouse model. ( A ) Gross morphology of the testis and epididymis from WT and cKO mice. ( B ) Comparison of the average sperm count in the cauda epididymis between the WT and cKO mice at 6-week-old age. Three biological repeats were performed. Data were shown as mean ± SD. ( C ) Representative Hematoxylin and Eosin (H E) staining of cauda epididymis and ( D ) phase-contrast microscopy of cauda sperm from WT and cKO littermates. Note that the majority of sperm exhibit deformed head morphology in the cKO cauda epididymis, as indicated. Data were presented as mean ± SEM.

    Techniques Used: Mouse Assay, Staining, Microscopy

    p300 methylation at the GBD domain attenuated the interaction between ACT and p300 in vitro and in vivo. ( A ) The effect of overexpression of CARM1 on the interaction between ACT and p300. CARM1 was induced for overexpression by doxycycline in the inducible CARM1 Flip-in HEK293 cell line. Reciprocal co-IP was performed as indicated using Flag- and GFP-tag antibodies, followed by immunoblotting with the indicated antibodies. ( B ) p300 is endogenously methylated at R2142 in the haploid spermatids. Endogenous p300 protein was immuno-precipitated with specific p300 antibody, followed by immunoblotting with various antibodies as indicated. Note that Carm1 cKO abolished the ADMA mark on the p300 protein, and more bound ACT protein was detected upon Carm1 cKO in the p300 immunoprecipitates. The heavy chain (HC) of IgG served as an antibody loading control. ( C ) Endogenous co-IP was carried out in the round spermatids purified from the WT and cKO testes. Note that elevated binding affinity was observed between ACT and p300 in the cKO spermatids. ( D ) A working model for the role of Carm1-mediated methylation in haploid spermatids development. In the early stage of haploid spermatids, ACT recruits p300 co-activators to activate germline-specific target gene expression. In the late stage of haploid spermatids, a cohort of CREMτ/ACT-bound target genes must be temporally inhibited through the disassembly of the p300•ACT•CREMτ axis by the methylation of p300 by Carm1, for the last wave of metabolic reprogramming in the elongating spermatids.
    Figure Legend Snippet: p300 methylation at the GBD domain attenuated the interaction between ACT and p300 in vitro and in vivo. ( A ) The effect of overexpression of CARM1 on the interaction between ACT and p300. CARM1 was induced for overexpression by doxycycline in the inducible CARM1 Flip-in HEK293 cell line. Reciprocal co-IP was performed as indicated using Flag- and GFP-tag antibodies, followed by immunoblotting with the indicated antibodies. ( B ) p300 is endogenously methylated at R2142 in the haploid spermatids. Endogenous p300 protein was immuno-precipitated with specific p300 antibody, followed by immunoblotting with various antibodies as indicated. Note that Carm1 cKO abolished the ADMA mark on the p300 protein, and more bound ACT protein was detected upon Carm1 cKO in the p300 immunoprecipitates. The heavy chain (HC) of IgG served as an antibody loading control. ( C ) Endogenous co-IP was carried out in the round spermatids purified from the WT and cKO testes. Note that elevated binding affinity was observed between ACT and p300 in the cKO spermatids. ( D ) A working model for the role of Carm1-mediated methylation in haploid spermatids development. In the early stage of haploid spermatids, ACT recruits p300 co-activators to activate germline-specific target gene expression. In the late stage of haploid spermatids, a cohort of CREMτ/ACT-bound target genes must be temporally inhibited through the disassembly of the p300•ACT•CREMτ axis by the methylation of p300 by Carm1, for the last wave of metabolic reprogramming in the elongating spermatids.

    Techniques Used: Methylation, Activated Clotting Time Assay, In Vitro, In Vivo, Over Expression, Co-Immunoprecipitation Assay, Purification, Binding Assay, Expressing

    15) Product Images from "Chromatin remodeling system p300-HDAC2-Sin3A is involved in Arginine Starvation-Induced HIF-1α Degradation at the ASS1 promoter for ASS1 Derepression"

    Article Title: Chromatin remodeling system p300-HDAC2-Sin3A is involved in Arginine Starvation-Induced HIF-1α Degradation at the ASS1 promoter for ASS1 Derepression

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-11445-0

    Effects of ADI on the regulation of HIF-1α stability by p300, HDAC2, and Sin3A. ( a ) ChIP assay of ASS1 promoter associations of p300, HDAC2 and Sin3A in A2058 cells treated with ADI for 15 min. ( b to d ) Effects of p300, Sin3A, and HDAC2 knockdown by siRNA as indicated on the expression of other proteins in the presence and absence of ADI (0.5 μg/ml, 1 hr). ( e to g ) ChIP assays of effects of p300, Sin3A, and HDAC2 knockdown on the ASS1 promoter associations of HIF1α, PHD2, HDAC2, p300, and Sin3A as indicated Cells were transfected with given siRNA as specified for 24 hr followed by ADI treatment (0.5 μg/ml) for 15 min.
    Figure Legend Snippet: Effects of ADI on the regulation of HIF-1α stability by p300, HDAC2, and Sin3A. ( a ) ChIP assay of ASS1 promoter associations of p300, HDAC2 and Sin3A in A2058 cells treated with ADI for 15 min. ( b to d ) Effects of p300, Sin3A, and HDAC2 knockdown by siRNA as indicated on the expression of other proteins in the presence and absence of ADI (0.5 μg/ml, 1 hr). ( e to g ) ChIP assays of effects of p300, Sin3A, and HDAC2 knockdown on the ASS1 promoter associations of HIF1α, PHD2, HDAC2, p300, and Sin3A as indicated Cells were transfected with given siRNA as specified for 24 hr followed by ADI treatment (0.5 μg/ml) for 15 min.

    Techniques Used: Chromatin Immunoprecipitation, Expressing, Transfection

    Effects of histone H3 deacetylation by ADI. ( a ) A2058 cells were treated with ADI for the time intervals as indicated, or with SAHA (20 μM for 1 hr as positive control), and lamin B expression (as control for sample loading). Acetylation status of various modified histones H3 were determined. ( b ) Reduction of ASS1-promoter associations of H3K27ac and H3K14ac by ADI (0.5 μg/ml, 15 min.). ( c to e ), Effects of p300, Sin3A, and HDAC2 knockdown by siRNAs, respectively, on the expression levels of H3K14ac and H3K27ac in A2058 cells treated with or without ADI. ( f ) Western blotting analyses of H3K14ac, H3K27ac, H3K9ac, and H3K18ac expression in A2058 and ADI-resistant (ADI R variants, 58R1 to 58R3). ( g ) ChIP assay of promoter-associations of p300, HDAC2, and HIF-1α in A2058 and ADI R cells (R1 to R3). ( h ) Western blotting analyses of HIF-1α, ASS1, H3K14ac, H3K27ac, and H3 expression in 4 matched pairs of primary cell lines derived from melanoma patients before (−) and after failed (+) by ADI treatments.
    Figure Legend Snippet: Effects of histone H3 deacetylation by ADI. ( a ) A2058 cells were treated with ADI for the time intervals as indicated, or with SAHA (20 μM for 1 hr as positive control), and lamin B expression (as control for sample loading). Acetylation status of various modified histones H3 were determined. ( b ) Reduction of ASS1-promoter associations of H3K27ac and H3K14ac by ADI (0.5 μg/ml, 15 min.). ( c to e ), Effects of p300, Sin3A, and HDAC2 knockdown by siRNAs, respectively, on the expression levels of H3K14ac and H3K27ac in A2058 cells treated with or without ADI. ( f ) Western blotting analyses of H3K14ac, H3K27ac, H3K9ac, and H3K18ac expression in A2058 and ADI-resistant (ADI R variants, 58R1 to 58R3). ( g ) ChIP assay of promoter-associations of p300, HDAC2, and HIF-1α in A2058 and ADI R cells (R1 to R3). ( h ) Western blotting analyses of HIF-1α, ASS1, H3K14ac, H3K27ac, and H3 expression in 4 matched pairs of primary cell lines derived from melanoma patients before (−) and after failed (+) by ADI treatments.

    Techniques Used: Positive Control, Expressing, Modification, Western Blot, Chromatin Immunoprecipitation, Derivative Assay

    Effects of ROS on ADI-induced HIF-1α degradation. ( a ) ADI-induced HF-1α degradation is inhibited by antioxidant, NAC. A2058 cells were treated with 10 μM MG-132 in the absence or presence of ADI or NAC (1 mM) for 4-h. Expression levels of HIF-1α, hydroxylated HIF-1α, and actin were determined by Western blotting. ( b ) The antioxidant NAC suppresses ADI-induced PHD2 enzymatic activity. A2058 cells were transfected with recombinant encoding HA-PHD2. Cells were treated with NAC or ADI as indicated for 1 hr. PHD2 enzymatic activity was measured using GST-ODDD (100 ng) as a substrate and production of HO-HIF-1α (p564). ( c ) Similar to those described in ( b ) was performed using anti-oxidants Mito-TEMPO (40 μM) and TEMPO (100 μM) for 1 hr. ( d , e ) Inhibitions of HDAC2 and PHD2 interaction by TEMPO in reciprocal co-IP assays. ( f , g ), Effects of antioxidants NAC (N, 1 mM) or TEMPO (T, 100 μM)) on ADI (A)-induced ASS1 promoter association of HIF-1 α , PHD2, p300, HDAC2 and H3K27ac in A2058 cells treated with or without ADI (A, 0.5 μg/ml, 1 hr) as determined by ChIP assay.
    Figure Legend Snippet: Effects of ROS on ADI-induced HIF-1α degradation. ( a ) ADI-induced HF-1α degradation is inhibited by antioxidant, NAC. A2058 cells were treated with 10 μM MG-132 in the absence or presence of ADI or NAC (1 mM) for 4-h. Expression levels of HIF-1α, hydroxylated HIF-1α, and actin were determined by Western blotting. ( b ) The antioxidant NAC suppresses ADI-induced PHD2 enzymatic activity. A2058 cells were transfected with recombinant encoding HA-PHD2. Cells were treated with NAC or ADI as indicated for 1 hr. PHD2 enzymatic activity was measured using GST-ODDD (100 ng) as a substrate and production of HO-HIF-1α (p564). ( c ) Similar to those described in ( b ) was performed using anti-oxidants Mito-TEMPO (40 μM) and TEMPO (100 μM) for 1 hr. ( d , e ) Inhibitions of HDAC2 and PHD2 interaction by TEMPO in reciprocal co-IP assays. ( f , g ), Effects of antioxidants NAC (N, 1 mM) or TEMPO (T, 100 μM)) on ADI (A)-induced ASS1 promoter association of HIF-1 α , PHD2, p300, HDAC2 and H3K27ac in A2058 cells treated with or without ADI (A, 0.5 μg/ml, 1 hr) as determined by ChIP assay.

    Techniques Used: Expressing, Western Blot, Activity Assay, Transfection, Recombinant, Co-Immunoprecipitation Assay, Chromatin Immunoprecipitation

    16) Product Images from "FANCJ protein is important for the stability of FANCD2/FANCI proteins and protects them from proteasome and caspase-3 dependent degradation"

    Article Title: FANCJ protein is important for the stability of FANCD2/FANCI proteins and protects them from proteasome and caspase-3 dependent degradation

    Journal: Oncotarget

    doi:

    Caspase-3 degrades FANCD2 in the absence of FANCJ and FANCJ helicase activity is not required for the stabilization of FANCD2 A. H1299 cells were transfected with control, FANCJ, and Caspase-3 siRNAs as indicated, and FANCD2 and FANCI proteins were examined by Western blots. B. The FANCJ-defective patient cell line EUFA30 was transfected with plasmids containing either wild-type or the helicase defective mutant (K52R) FANCJ. The effects of these two FANCJ variants on FANCD2 stability were determined by Western blot analysis. C. Protein extracts from H1299 cells expressing either a wild-type or helicase-defective mutant (K52R) FANCJ (both labeled with a Myc-tag) were selected to co-immunoprecipitation with antibodies directed against either FANCD2 or the Myc-tag on the 2 FANCJ variants (WT and K52R). The precipitated proteins were blotted for the corresponding partner protein (FANCD2 or Myc). FANCD2 was able to pull down both wild-type and the helicase-deficient FANCJ and FANCD2 in turn were found in immunoprecipations of both FANCJs.
    Figure Legend Snippet: Caspase-3 degrades FANCD2 in the absence of FANCJ and FANCJ helicase activity is not required for the stabilization of FANCD2 A. H1299 cells were transfected with control, FANCJ, and Caspase-3 siRNAs as indicated, and FANCD2 and FANCI proteins were examined by Western blots. B. The FANCJ-defective patient cell line EUFA30 was transfected with plasmids containing either wild-type or the helicase defective mutant (K52R) FANCJ. The effects of these two FANCJ variants on FANCD2 stability were determined by Western blot analysis. C. Protein extracts from H1299 cells expressing either a wild-type or helicase-defective mutant (K52R) FANCJ (both labeled with a Myc-tag) were selected to co-immunoprecipitation with antibodies directed against either FANCD2 or the Myc-tag on the 2 FANCJ variants (WT and K52R). The precipitated proteins were blotted for the corresponding partner protein (FANCD2 or Myc). FANCD2 was able to pull down both wild-type and the helicase-deficient FANCJ and FANCD2 in turn were found in immunoprecipations of both FANCJs.

    Techniques Used: Activity Assay, Transfection, Western Blot, Mutagenesis, Expressing, Labeling, Immunoprecipitation

    Proposed model for FANCJ stabilization of FANCD2 and FANCI FANCJ exists in a complex with both FANCD2 and FANCI in undamaged cells. This complex exists principally in the cytoplasm, but is also present in the nucleus. When there is DNA damage FANCD2 can be activated by the FA core complex and transmitted to the nucleus to participate in the formation of repair foci at the site of damage. When the K52R helicase-dead mutant, and potentially other FANCJ point mutations, is part of the complex it would protect FANCD2 but may still result in decreased DNA repair. For example, the K52R mutation has previously been shown to increase sensitivity to certain types of DNA damage, such as ionizing radiation [ 48 ], suggesting FANCJ helicase functions could be independent from stabilizing FANCD2/FANI proteins. When FANCJ is lost the unprotected FANCD2 and FANCI proteins are degraded by the ubiquitin proteasome and caspase-3.
    Figure Legend Snippet: Proposed model for FANCJ stabilization of FANCD2 and FANCI FANCJ exists in a complex with both FANCD2 and FANCI in undamaged cells. This complex exists principally in the cytoplasm, but is also present in the nucleus. When there is DNA damage FANCD2 can be activated by the FA core complex and transmitted to the nucleus to participate in the formation of repair foci at the site of damage. When the K52R helicase-dead mutant, and potentially other FANCJ point mutations, is part of the complex it would protect FANCD2 but may still result in decreased DNA repair. For example, the K52R mutation has previously been shown to increase sensitivity to certain types of DNA damage, such as ionizing radiation [ 48 ], suggesting FANCJ helicase functions could be independent from stabilizing FANCD2/FANI proteins. When FANCJ is lost the unprotected FANCD2 and FANCI proteins are degraded by the ubiquitin proteasome and caspase-3.

    Techniques Used: Mutagenesis

    FANCJ regulates FANCD2 stability, but FANCD2 has little to no effect on FANCJ protein levels A. HDF or B. H1299 cells were transfected with siRNAs for either FANCJ or FANCD2 and the levels of FANCJ, FANCD2, and FANCI were measured by Western blot. C. The average values of FANCJ, FANCD2, and FANCI in cells treated with siRNAs for FANCJ or FANCD2, from at least three independent experiments, were normalized to the levels in H1299 cells treated with control siRNA. Bars represent standard error from multiple (3–4) independent experiments. * indicates the value is significantly altered from control ( P
    Figure Legend Snippet: FANCJ regulates FANCD2 stability, but FANCD2 has little to no effect on FANCJ protein levels A. HDF or B. H1299 cells were transfected with siRNAs for either FANCJ or FANCD2 and the levels of FANCJ, FANCD2, and FANCI were measured by Western blot. C. The average values of FANCJ, FANCD2, and FANCI in cells treated with siRNAs for FANCJ or FANCD2, from at least three independent experiments, were normalized to the levels in H1299 cells treated with control siRNA. Bars represent standard error from multiple (3–4) independent experiments. * indicates the value is significantly altered from control ( P

    Techniques Used: Transfection, Western Blot

    Down-regulation or loss of FANCJ concomitantly diminishes FANCD2 and FANCI proteins in multiple cell lines HDF A. , A549 B. , H1299 C. , OV90 D. and SKVO3 E. cells were transfected with control or FANCJ siRNAs. After 48 hours whole cell lysates were collected, normalized for total protein concentration, and assessed for the levels of FANCD2, FANCI, FANCJ, and GAPDH proteins by Western blotting. F. A second, previously validated, FANCJ siRNA (FANCJ-2) was used to verify that the effects were not the result of non-specific interactions of the original siRNA.
    Figure Legend Snippet: Down-regulation or loss of FANCJ concomitantly diminishes FANCD2 and FANCI proteins in multiple cell lines HDF A. , A549 B. , H1299 C. , OV90 D. and SKVO3 E. cells were transfected with control or FANCJ siRNAs. After 48 hours whole cell lysates were collected, normalized for total protein concentration, and assessed for the levels of FANCD2, FANCI, FANCJ, and GAPDH proteins by Western blotting. F. A second, previously validated, FANCJ siRNA (FANCJ-2) was used to verify that the effects were not the result of non-specific interactions of the original siRNA.

    Techniques Used: Transfection, Protein Concentration, Western Blot

    17) Product Images from "Temporal and spatial characterization of nonsense-mediated mRNA decay"

    Article Title: Temporal and spatial characterization of nonsense-mediated mRNA decay

    Journal: Genes & Development

    doi: 10.1101/gad.209635.112

    The steady-state level of cytoplasmic Gl TER mRNA depends on PTC recognition and UPF1 and is resistant to NMD. ( A) U2OS Tet-On cells were transfected with the bidirectional promoter plasmid expressing GI TER and NORM mRNAs alone or with a plasmid expressing
    Figure Legend Snippet: The steady-state level of cytoplasmic Gl TER mRNA depends on PTC recognition and UPF1 and is resistant to NMD. ( A) U2OS Tet-On cells were transfected with the bidirectional promoter plasmid expressing GI TER and NORM mRNAs alone or with a plasmid expressing

    Techniques Used: Transfection, Plasmid Preparation, Expressing

    18) Product Images from "Cyclin C is a haploinsufficient tumor suppressor"

    Article Title: Cyclin C is a haploinsufficient tumor suppressor

    Journal: Nature cell biology

    doi: 10.1038/ncb3046

    Phosphorylation of ICN1 by cyclin C-CDK8, C-CDK19 and C-CDK3 kinases. ( a ) Cyclin C was immunoprecipitated (IP) from MOLT-16 cells, immunoblots were probed with the indicated antibodies. Lysate, whole cell extracts. ( b ) Cyclin C F/F MEFs stably expressing ICN1 were transduced with two anti-CDK8 shRNAs (1 or 2) or with control shRNA (CTR). Cell extracts were imunoblotted with the indicated antibodies. Notch1, antibody against full-length Notch1. ( c ) Similar analysis as in b , using anti-CDK19 shRNAs (1, 2 or 3). ( d ), Purified, recombinant ICN1 was incubated in the presence (+) or absence (−) of recombinant cyclin C-CDK8 with γ[ 32 P]ATP. Upper panel: proteins analyzed by autoradiography, 32 P-ICN1 denotes phosphorylated ICN1. Lower panel: ICN1 visualized by Ponceau S staining. ( e ) His-tagged cyclin C or cyclin C and CDK19 were expressed in Sf9 cells, purified using anti-His antibodies, and used for in vitro kinase reactions in the presence (+) or absence (−) of ICN1 as a substrate with γ[ 32 P]ATP. As a positive control, ICN1 was incubated with recombinant cyclin C-CDK8. Upper panel: proteins analyzed by autoradiography to detect phosphorylated ICN1 ( 32 P-ICN1). Second panel: GST-ICN1 protein detected by immunoblotting with anti-GST antibody. Third and fourth panels: CDK19 and cyclin C detected using the indicated antibodies. ( f ) Similar analyses as in e ,using cyclin C and CDK3. ICN1 protein was detected by Ponceau S staining. ( g ) A diagram of ICN1 protein used for phosphorylation analyses, with RAM (RBP-Jkappa-associated-module), Ankyrin repeat, transactivation domain (TAD) and PEST domains marked. Numbers above the ICN1 depict residues identified to be phosphorylated by cyclin C-CDK8 in vitro , numbers below show residues phosphorylated in vivo . ( h ) Protein extracts were prepared from thymocytes from C F/F /Mx1-Cre − (Ctrl) and C Δ/Δ /Mx1-Cre (C-KO) mice, ICN1 was immunoprecipitated (IP) and immunoblots probed with a phospho-specific antibody against Ser2517-phoshorylated ICN1 (phospho-ICN1), or with anti-ICN1 antibody. Input denotes whole cell extracts. ( i ) Purified recombinant GST-ICN1 was pre-incubated in the presence (+) or absence (−) of cyclin C-CDK8 kinase. Subsequently, GST-ICN1 was incubated with in vitro translated 35 S-labeled Fbw7. ICN1-bound proteins were visualized by autoradiography. GST beads were used as a negative control. Input: an aliquot of radiolabeled Fbw7. Note that ICN1 interacted with Fbw7 only after pre-incubation with cyclin C-CDK8. Lower panel: Coomassie stained gel. The middle portion of the gel was cut out and the images were spliced together (dashed line). ( j ) Protein extracts were prepared from thymocytes from Ctrl and C-KO mice (as in h ). Polyubiquitinated proteins were captured with Halo-TUBE (AP, affinity purification poly-ubiquitin), and immunoblotted with the indicated antibodies. Input, cell extracts. Note reduced polyubiquitination of the endogenous ICN1 in C-KO thymocytes.
    Figure Legend Snippet: Phosphorylation of ICN1 by cyclin C-CDK8, C-CDK19 and C-CDK3 kinases. ( a ) Cyclin C was immunoprecipitated (IP) from MOLT-16 cells, immunoblots were probed with the indicated antibodies. Lysate, whole cell extracts. ( b ) Cyclin C F/F MEFs stably expressing ICN1 were transduced with two anti-CDK8 shRNAs (1 or 2) or with control shRNA (CTR). Cell extracts were imunoblotted with the indicated antibodies. Notch1, antibody against full-length Notch1. ( c ) Similar analysis as in b , using anti-CDK19 shRNAs (1, 2 or 3). ( d ), Purified, recombinant ICN1 was incubated in the presence (+) or absence (−) of recombinant cyclin C-CDK8 with γ[ 32 P]ATP. Upper panel: proteins analyzed by autoradiography, 32 P-ICN1 denotes phosphorylated ICN1. Lower panel: ICN1 visualized by Ponceau S staining. ( e ) His-tagged cyclin C or cyclin C and CDK19 were expressed in Sf9 cells, purified using anti-His antibodies, and used for in vitro kinase reactions in the presence (+) or absence (−) of ICN1 as a substrate with γ[ 32 P]ATP. As a positive control, ICN1 was incubated with recombinant cyclin C-CDK8. Upper panel: proteins analyzed by autoradiography to detect phosphorylated ICN1 ( 32 P-ICN1). Second panel: GST-ICN1 protein detected by immunoblotting with anti-GST antibody. Third and fourth panels: CDK19 and cyclin C detected using the indicated antibodies. ( f ) Similar analyses as in e ,using cyclin C and CDK3. ICN1 protein was detected by Ponceau S staining. ( g ) A diagram of ICN1 protein used for phosphorylation analyses, with RAM (RBP-Jkappa-associated-module), Ankyrin repeat, transactivation domain (TAD) and PEST domains marked. Numbers above the ICN1 depict residues identified to be phosphorylated by cyclin C-CDK8 in vitro , numbers below show residues phosphorylated in vivo . ( h ) Protein extracts were prepared from thymocytes from C F/F /Mx1-Cre − (Ctrl) and C Δ/Δ /Mx1-Cre (C-KO) mice, ICN1 was immunoprecipitated (IP) and immunoblots probed with a phospho-specific antibody against Ser2517-phoshorylated ICN1 (phospho-ICN1), or with anti-ICN1 antibody. Input denotes whole cell extracts. ( i ) Purified recombinant GST-ICN1 was pre-incubated in the presence (+) or absence (−) of cyclin C-CDK8 kinase. Subsequently, GST-ICN1 was incubated with in vitro translated 35 S-labeled Fbw7. ICN1-bound proteins were visualized by autoradiography. GST beads were used as a negative control. Input: an aliquot of radiolabeled Fbw7. Note that ICN1 interacted with Fbw7 only after pre-incubation with cyclin C-CDK8. Lower panel: Coomassie stained gel. The middle portion of the gel was cut out and the images were spliced together (dashed line). ( j ) Protein extracts were prepared from thymocytes from Ctrl and C-KO mice (as in h ). Polyubiquitinated proteins were captured with Halo-TUBE (AP, affinity purification poly-ubiquitin), and immunoblotted with the indicated antibodies. Input, cell extracts. Note reduced polyubiquitination of the endogenous ICN1 in C-KO thymocytes.

    Techniques Used: Immunoprecipitation, Western Blot, Stable Transfection, Expressing, Transduction, shRNA, Purification, Recombinant, Incubation, Autoradiography, Staining, In Vitro, Positive Control, In Vivo, Mouse Assay, Labeling, Negative Control, Affinity Purification

    T-ALL patient-derived mutations block the ability of cyclin C-CDK to phosphorylate critical ICN1 residues and are oncogenic  in vivo.  ( a ) Schematic representation of missense mutations found in T-ALL patients. ICN1 aminoacids shown in red represent critical cyclin C-CDK-dependent phosphoresidues on Notch1 (T2512, S2514, S2517). Arrowheads depict mutations found in T-ALL patients. All 5 patients had also mutations within Notch1 HD-domain. ( b ) Mass spectrometry quantitative analysis of ICN1 phosphorylation levels at three critical cyclin C-CDK8-dependent phosphoresidues. A peptide containing wild-type ICN1 sequence, or peptides with the indicated patient-derived aminoacid substitutions were incubated with cyclin C-CDK8 kinase, and stoichiometry of phosphorylation at T2512, S2514 and S2517 was quantified by mass spectrometry. The level of phosphorylation seen in ICN1 wild-type peptide was set at 100%. ( c ) Human T-ALL MOLT-4 cells were transduced with retroviruses expressing Myc-tagged wild-type ICN1 (WT) plus GFP, or ICN1 containing patient-derived P2515R mutation plus GFP, or GFP only. ICN1 was immunoprecipitated from GFP +  cells using anti-Myc antibody, immunoblots were probed with indicated antibodies. Phospho-ICN1 denotes anti-phospho-Ser2517-ICN1 antibody. ( d ) Purified, recombinant GST-tagged wild-type ICN1, or ICN1 mutants containing the indicated patient-derived aminoacid substitutions were pre-incubated with cyclin C-CDK8 kinase. Subsequently, GST-ICN1 was incubated with  in vitro  translated  35 S-labeled Fbw7. ICN1-bound Fbw7 was visualized by autoradiography. Input: an aliquot of radiolabeled Fbw7. Note that all patient-derived mutants were compromised in their ability to bind Fbw7. Lower panel: Coomassie-stained gel. The middle portion of the gel was cut out and images were spliced together (dashed line). ( e ) HeLa cells were transfected with an empty vector (EV), Myc-tagged wild-type ICN1 (WT) or ICN1 mutants containing the indicated patient-derived aminoacid substitutions, together with HA-tagged-Fbw7. Fbw7 was immunoprecipitated (IP HA), immunoblots were probed with anti-ICN1 antibody (to visualize Fbw7-bound ICN1). Note that all patient-derived mutants were compromised in their ability to bind Fbw7. Input, whole cell lysates. f , Mice received wild-type HPC transduced with Notch1-P12 (n=7), Notch1-P12 carrying patient-derived S2514F mutation (n=9) or Notch1-P12 with P2513A, S2514F and P2515R mutations (n=6). Shown is Kaplan-Meier analysis of survival.  P =0.03 for Notch1-P12 WT vs. Notch1-P12 S2514F;  P =0.01 for Notch1-P12 WT vs. Notch1-P12 P2513A/S2514F/P2515R (Log-rank test).
    Figure Legend Snippet: T-ALL patient-derived mutations block the ability of cyclin C-CDK to phosphorylate critical ICN1 residues and are oncogenic in vivo. ( a ) Schematic representation of missense mutations found in T-ALL patients. ICN1 aminoacids shown in red represent critical cyclin C-CDK-dependent phosphoresidues on Notch1 (T2512, S2514, S2517). Arrowheads depict mutations found in T-ALL patients. All 5 patients had also mutations within Notch1 HD-domain. ( b ) Mass spectrometry quantitative analysis of ICN1 phosphorylation levels at three critical cyclin C-CDK8-dependent phosphoresidues. A peptide containing wild-type ICN1 sequence, or peptides with the indicated patient-derived aminoacid substitutions were incubated with cyclin C-CDK8 kinase, and stoichiometry of phosphorylation at T2512, S2514 and S2517 was quantified by mass spectrometry. The level of phosphorylation seen in ICN1 wild-type peptide was set at 100%. ( c ) Human T-ALL MOLT-4 cells were transduced with retroviruses expressing Myc-tagged wild-type ICN1 (WT) plus GFP, or ICN1 containing patient-derived P2515R mutation plus GFP, or GFP only. ICN1 was immunoprecipitated from GFP + cells using anti-Myc antibody, immunoblots were probed with indicated antibodies. Phospho-ICN1 denotes anti-phospho-Ser2517-ICN1 antibody. ( d ) Purified, recombinant GST-tagged wild-type ICN1, or ICN1 mutants containing the indicated patient-derived aminoacid substitutions were pre-incubated with cyclin C-CDK8 kinase. Subsequently, GST-ICN1 was incubated with in vitro translated 35 S-labeled Fbw7. ICN1-bound Fbw7 was visualized by autoradiography. Input: an aliquot of radiolabeled Fbw7. Note that all patient-derived mutants were compromised in their ability to bind Fbw7. Lower panel: Coomassie-stained gel. The middle portion of the gel was cut out and images were spliced together (dashed line). ( e ) HeLa cells were transfected with an empty vector (EV), Myc-tagged wild-type ICN1 (WT) or ICN1 mutants containing the indicated patient-derived aminoacid substitutions, together with HA-tagged-Fbw7. Fbw7 was immunoprecipitated (IP HA), immunoblots were probed with anti-ICN1 antibody (to visualize Fbw7-bound ICN1). Note that all patient-derived mutants were compromised in their ability to bind Fbw7. Input, whole cell lysates. f , Mice received wild-type HPC transduced with Notch1-P12 (n=7), Notch1-P12 carrying patient-derived S2514F mutation (n=9) or Notch1-P12 with P2513A, S2514F and P2515R mutations (n=6). Shown is Kaplan-Meier analysis of survival. P =0.03 for Notch1-P12 WT vs. Notch1-P12 S2514F; P =0.01 for Notch1-P12 WT vs. Notch1-P12 P2513A/S2514F/P2515R (Log-rank test).

    Techniques Used: Derivative Assay, Blocking Assay, In Vivo, Mass Spectrometry, Sequencing, Incubation, Transduction, Expressing, Mutagenesis, Immunoprecipitation, Western Blot, Purification, Recombinant, In Vitro, Labeling, Autoradiography, Staining, Transfection, Plasmid Preparation, Mouse Assay

    Cyclin C is heterozygously deleted in human T-ALL. ( a ) Array CGH was performed on genomic DNA from primary T-ALL patient samples and ( b ) human T-ALL cell lines. Patient samples are grouped by clinical outcome (IF, induction failure; REL, relapse; EFS, long-term event-free survivor). Shown is a dChip plot of the segmented CGH log2 copy number ratios of the entire chromosome 6. Location of the CCNC locus is denoted by a red line. ( c ) Cyclin C expression was analyzed using gene expression microarrays; analysis was performed on a subset of primary T-ALL patient samples depicted in a . Shown are cyclin C expression levels in tumors which displayed heterozygous CCNC gene deletions (n=6) vs. tumors without CCNC deletion ( CCNC no del, n=34). P =0.006 (Mann-Whitney test).
    Figure Legend Snippet: Cyclin C is heterozygously deleted in human T-ALL. ( a ) Array CGH was performed on genomic DNA from primary T-ALL patient samples and ( b ) human T-ALL cell lines. Patient samples are grouped by clinical outcome (IF, induction failure; REL, relapse; EFS, long-term event-free survivor). Shown is a dChip plot of the segmented CGH log2 copy number ratios of the entire chromosome 6. Location of the CCNC locus is denoted by a red line. ( c ) Cyclin C expression was analyzed using gene expression microarrays; analysis was performed on a subset of primary T-ALL patient samples depicted in a . Shown are cyclin C expression levels in tumors which displayed heterozygous CCNC gene deletions (n=6) vs. tumors without CCNC deletion ( CCNC no del, n=34). P =0.006 (Mann-Whitney test).

    Techniques Used: Expressing, MANN-WHITNEY

    Gene expression analyses of cyclin C-null cells. ( a ) CDK8 was immunoprecipitated (IP) from wild-type (WT) or cyclin C Δ/Δ (C-KO) MEFs and used for in vitro kinase reactions with recombinant carboxy terminal domain (CTD) of RNA polymerase II as a substrate, in the presence of γ[ 32 P]ATP. C-KO + cyclin C: cyclin C Δ/Δ MEFs engineered to ectopically express cyclin C. Note that re-expression of cyclin C restored CDK8 kinase activity in cyclin C Δ/Δ cells. IgG was used for control immunoprecipitation. 32 P-CTD denotes phosphorylated CTD, detected by autoradiography. Lower panel, CTD was visualized by Coomassie staining. ( b ) Lysates from wild-type (WT), cyclin C +/Δ (C-HET) and cyclin C Δ/Δ (C-KO) ES cells were probed with an antibody against phospho-Ser 2 and phospho-Ser 5 of RNA polymerase II CTD. Lower panel: immunoblotting with an anti-CTD antibody. ( c ) Cyclin C, CDK8, MED12 or MED13 were immunoprecipitated (IP) from wild-type or C-KO MEFs; the immunoblots were probed with the indicated antibodies, along with whole cell extracts (Input). ( d ) Scatterplot showing log of the normalized gene expression in control (x-axis) vs. C Δ/Δ (y-axis) MEFs, ES cells (ESC) and E18.5 brains. Parallel lines indicate 2-fold change (up- or down-regulation) in transcript levels. ( e ) Scatterplot showing log of the normalized gene expression values in control brains (x-axis) vs. control ES cells (y-axis). This is a control to panel d . It illustrates that different compartments (brain, ES cells) display very distinct gene expression patterns, as expected.
    Figure Legend Snippet: Gene expression analyses of cyclin C-null cells. ( a ) CDK8 was immunoprecipitated (IP) from wild-type (WT) or cyclin C Δ/Δ (C-KO) MEFs and used for in vitro kinase reactions with recombinant carboxy terminal domain (CTD) of RNA polymerase II as a substrate, in the presence of γ[ 32 P]ATP. C-KO + cyclin C: cyclin C Δ/Δ MEFs engineered to ectopically express cyclin C. Note that re-expression of cyclin C restored CDK8 kinase activity in cyclin C Δ/Δ cells. IgG was used for control immunoprecipitation. 32 P-CTD denotes phosphorylated CTD, detected by autoradiography. Lower panel, CTD was visualized by Coomassie staining. ( b ) Lysates from wild-type (WT), cyclin C +/Δ (C-HET) and cyclin C Δ/Δ (C-KO) ES cells were probed with an antibody against phospho-Ser 2 and phospho-Ser 5 of RNA polymerase II CTD. Lower panel: immunoblotting with an anti-CTD antibody. ( c ) Cyclin C, CDK8, MED12 or MED13 were immunoprecipitated (IP) from wild-type or C-KO MEFs; the immunoblots were probed with the indicated antibodies, along with whole cell extracts (Input). ( d ) Scatterplot showing log of the normalized gene expression in control (x-axis) vs. C Δ/Δ (y-axis) MEFs, ES cells (ESC) and E18.5 brains. Parallel lines indicate 2-fold change (up- or down-regulation) in transcript levels. ( e ) Scatterplot showing log of the normalized gene expression values in control brains (x-axis) vs. control ES cells (y-axis). This is a control to panel d . It illustrates that different compartments (brain, ES cells) display very distinct gene expression patterns, as expected.

    Techniques Used: Expressing, Immunoprecipitation, In Vitro, Recombinant, Activity Assay, Autoradiography, Staining, Western Blot

    Cell cycle analyses of cyclin C-null cells. ( a ) In vitro growth-curves of cyclin C F/F (Ctrl) and C Δ/Δ (C-KO) MEFs, n=3. P =0.8 (day 4), P =0.8 (day 5), P =0.7 (day 6, all using t-test). ( b ) Fraction of cyclin C F/F (Ctrl) and C Δ/Δ (C-KO) MEFs in different cell cycle phases, visualized by propidium iodide (DNA content) and anti-BrdU staining followed by FACS. ( c ) MEFs were rendered quiescent by serum starvation, and then stimulated to re-enter the cell cycle from G 0 by addition of medium containing the indicated concentrations of serum (FCS). Entry of cells into the S-phase was evaluated by measuring [ 3 H]-thymidine uptake. Left panel: P =0.003 (at 18 hrs); middle panel: P =0.028 (at 18 hrs); right panel: P =0.007 (C Δ/Δ vs. C Δ/Δ Rb Δ/Δ ), P =0.202 (Rb Δ/Δ vs. C Δ/Δ Rb Δ/Δ ), both at 12 hrs. P values were determined by t-test. ( d ) Wild-type MEFs were arrested in G 0 as above, and then stimulated to re-enter the cell cycle by addition of medium containing 10% serum. Upper panel: at the indicated time-points, cyclin C was immunoprecipitated and used for in vitro kinase reactions with recombinant pRB as a substrate in the presence of γ[ 32 P]ATP. 32 P-pRB denotes phosphorylated pRB, detected by autoradiography. Lower panel: the levels of cyclin C at the indicated time-points were determined by western blotting. ( e ) CDK1 or CDK2 were immunoprecipitated from quiescent MEFs (0h), or at 6 hrs after serum stimulation, or from asynchronously growing cells (Cycling). Immunoblots were probed with the indicated antibodies. Lysate, denotes straight lysate from cycling cells. ( f ) Flag-tagged CDK2 was expressed in 293T cells and immunoprecipitated (IP) with an anti-Flag antibody. Complexes were eluted with a Flag peptide, re-immunoprecipitated (re-IP) with an anti-cyclin C antibody (or, for control with IgG), and used for in vitro kinase reactions with recombinant pRB as a substrate. 32 P-pRB denotes phosphorylated pRB, detected by autoradiography.
    Figure Legend Snippet: Cell cycle analyses of cyclin C-null cells. ( a ) In vitro growth-curves of cyclin C F/F (Ctrl) and C Δ/Δ (C-KO) MEFs, n=3. P =0.8 (day 4), P =0.8 (day 5), P =0.7 (day 6, all using t-test). ( b ) Fraction of cyclin C F/F (Ctrl) and C Δ/Δ (C-KO) MEFs in different cell cycle phases, visualized by propidium iodide (DNA content) and anti-BrdU staining followed by FACS. ( c ) MEFs were rendered quiescent by serum starvation, and then stimulated to re-enter the cell cycle from G 0 by addition of medium containing the indicated concentrations of serum (FCS). Entry of cells into the S-phase was evaluated by measuring [ 3 H]-thymidine uptake. Left panel: P =0.003 (at 18 hrs); middle panel: P =0.028 (at 18 hrs); right panel: P =0.007 (C Δ/Δ vs. C Δ/Δ Rb Δ/Δ ), P =0.202 (Rb Δ/Δ vs. C Δ/Δ Rb Δ/Δ ), both at 12 hrs. P values were determined by t-test. ( d ) Wild-type MEFs were arrested in G 0 as above, and then stimulated to re-enter the cell cycle by addition of medium containing 10% serum. Upper panel: at the indicated time-points, cyclin C was immunoprecipitated and used for in vitro kinase reactions with recombinant pRB as a substrate in the presence of γ[ 32 P]ATP. 32 P-pRB denotes phosphorylated pRB, detected by autoradiography. Lower panel: the levels of cyclin C at the indicated time-points were determined by western blotting. ( e ) CDK1 or CDK2 were immunoprecipitated from quiescent MEFs (0h), or at 6 hrs after serum stimulation, or from asynchronously growing cells (Cycling). Immunoblots were probed with the indicated antibodies. Lysate, denotes straight lysate from cycling cells. ( f ) Flag-tagged CDK2 was expressed in 293T cells and immunoprecipitated (IP) with an anti-Flag antibody. Complexes were eluted with a Flag peptide, re-immunoprecipitated (re-IP) with an anti-cyclin C antibody (or, for control with IgG), and used for in vitro kinase reactions with recombinant pRB as a substrate. 32 P-pRB denotes phosphorylated pRB, detected by autoradiography.

    Techniques Used: In Vitro, BrdU Staining, FACS, Immunoprecipitation, Recombinant, Autoradiography, Western Blot

    Genetic ablation of cyclin C upregulates ICN1. ( a ) Appearance of thymuses from 6-weeks-old control (C F/F /Mx1-Cre − ) and C Δ/Δ /Mx1-Cre (C-KO) mice. Scale bar, 4 mm. ( b ) Total number of thymocytes in 4-6 weeks-old control (n=10) and C-KO mice (n=10). Horizontal lines denote mean values. P
    Figure Legend Snippet: Genetic ablation of cyclin C upregulates ICN1. ( a ) Appearance of thymuses from 6-weeks-old control (C F/F /Mx1-Cre − ) and C Δ/Δ /Mx1-Cre (C-KO) mice. Scale bar, 4 mm. ( b ) Total number of thymocytes in 4-6 weeks-old control (n=10) and C-KO mice (n=10). Horizontal lines denote mean values. P

    Techniques Used: Mouse Assay

    Cyclin C is a haploinsufficient tumor suppressor. ( a ) Tumor incidence (left) and Kaplan-Meier survival analysis (right) in C F/F /LMO1/Mx-Cre − (Ctrl) and C Δ/Δ /LMO1/Mx-Cre + (C-KO) mice. N=14 per group. P =0.0023 (Log-rank test). ( b ) Tumor sections (genotypes as in a ) were stained for ICN1. Note increased ICN1 levels in C-KO tumor. Human T-ALL KOPT-K1 cells were used as a positive control. Scale bar, 100μm ( c ) Schematic representation of the experimental design to trigger T-ALL in mice. C F/F /Mx1-Cre − (Ctrl), C +/F /Mx1-Cre (C-HET), or C F/F /Mx1-Cre (C-KO) animals were injected with poIy-polyC (to delete cyclin C). Bone marrow was collected, HPC were sorted, and transduced with a retrovirus encoding ICN1 and GFP, or Notch1-P12 and GFP and injected into wild-type recipient mice. ( d ) HPC were isolated from pI-pC-treated C F/F /Mx1-Cre − (Ctrl), C +/Δ /Mx1-Cre (C-HET) and C Δ/Δ /Mx1-Cre (C-KO) animals and cultured for 48 h on OP9-DL1 stroma. The levels of ICN1 and cyclin C were detected by immunoblotting. Note increased endogenous ICN1 levels in C-HET and C-KO. ( e ) Comparison of ICN1 levels in non-manipulated HPC from C-KO mice (C-KO, −Notch1), vs. HPC from control mice transduced with oncogenic Notch1-P12 (Ctrl, +Notch1). Note strongly increased ( > 6-fold) ICN1 levels in cells expressing Notch1-P12. Hence, although cyclin C-KO HPC express elevated levels of endogenous ICN1 as compared to control HPC (panel d ), this level is still substantially lower than the level of ectopically expressed ICN1 upon viral transduction (i.e., the level needed to trigger T-ALL). ( f ) Comparison of ICN1 levels in HPC of Ctrl vs. C-KO mice. Both genotypes were transduced with Notch1-P12. Note elevated (~4-fold) levels of ectopically expressed ICN1 in C-KO cells. The middle portion of the gel was cut out and the images were spliced together (dashed line). ( g ) Comparison of ICN1 levels in HPC of Ctrl vs. C-HET mice. Both genotypes were transduced with Notch1-P12. Note elevated (~2-fold) levels of ectopically expressed ICN1 in C-HET cells. In e-g , numbers below the gels denote densitometric quantification of ICN1 band densities (ImageJ). Dashed lines indicate that the lane 2 in panel e , lane 1 in panel f , and lane 1 in panel g show control HPC transduced with Notch1-P12. ( h ) Mice received ICN1-transduced C F/F (Ctrl) or C Δ/Δ (C-KO) HPC. Left: tumor incidence, n=20 per group. Right: Kaplan-Meier analysis of survival, n=14 in control, n=10 in cyclin C-KO. P
    Figure Legend Snippet: Cyclin C is a haploinsufficient tumor suppressor. ( a ) Tumor incidence (left) and Kaplan-Meier survival analysis (right) in C F/F /LMO1/Mx-Cre − (Ctrl) and C Δ/Δ /LMO1/Mx-Cre + (C-KO) mice. N=14 per group. P =0.0023 (Log-rank test). ( b ) Tumor sections (genotypes as in a ) were stained for ICN1. Note increased ICN1 levels in C-KO tumor. Human T-ALL KOPT-K1 cells were used as a positive control. Scale bar, 100μm ( c ) Schematic representation of the experimental design to trigger T-ALL in mice. C F/F /Mx1-Cre − (Ctrl), C +/F /Mx1-Cre (C-HET), or C F/F /Mx1-Cre (C-KO) animals were injected with poIy-polyC (to delete cyclin C). Bone marrow was collected, HPC were sorted, and transduced with a retrovirus encoding ICN1 and GFP, or Notch1-P12 and GFP and injected into wild-type recipient mice. ( d ) HPC were isolated from pI-pC-treated C F/F /Mx1-Cre − (Ctrl), C +/Δ /Mx1-Cre (C-HET) and C Δ/Δ /Mx1-Cre (C-KO) animals and cultured for 48 h on OP9-DL1 stroma. The levels of ICN1 and cyclin C were detected by immunoblotting. Note increased endogenous ICN1 levels in C-HET and C-KO. ( e ) Comparison of ICN1 levels in non-manipulated HPC from C-KO mice (C-KO, −Notch1), vs. HPC from control mice transduced with oncogenic Notch1-P12 (Ctrl, +Notch1). Note strongly increased ( > 6-fold) ICN1 levels in cells expressing Notch1-P12. Hence, although cyclin C-KO HPC express elevated levels of endogenous ICN1 as compared to control HPC (panel d ), this level is still substantially lower than the level of ectopically expressed ICN1 upon viral transduction (i.e., the level needed to trigger T-ALL). ( f ) Comparison of ICN1 levels in HPC of Ctrl vs. C-KO mice. Both genotypes were transduced with Notch1-P12. Note elevated (~4-fold) levels of ectopically expressed ICN1 in C-KO cells. The middle portion of the gel was cut out and the images were spliced together (dashed line). ( g ) Comparison of ICN1 levels in HPC of Ctrl vs. C-HET mice. Both genotypes were transduced with Notch1-P12. Note elevated (~2-fold) levels of ectopically expressed ICN1 in C-HET cells. In e-g , numbers below the gels denote densitometric quantification of ICN1 band densities (ImageJ). Dashed lines indicate that the lane 2 in panel e , lane 1 in panel f , and lane 1 in panel g show control HPC transduced with Notch1-P12. ( h ) Mice received ICN1-transduced C F/F (Ctrl) or C Δ/Δ (C-KO) HPC. Left: tumor incidence, n=20 per group. Right: Kaplan-Meier analysis of survival, n=14 in control, n=10 in cyclin C-KO. P

    Techniques Used: Mouse Assay, Staining, Positive Control, Injection, Transduction, Isolation, Cell Culture, Expressing

    Generation and analyses of cyclin C knockout mice. ( a ) Cyclin C gene targeting strategy. Coding exons are shown as filled boxes. Neo, neomycin phosphotransferase gene; loxP and FRT sequences are indicated as light blue triangles and dark blue rectangles, respectively. Restriction enzymes recognition sites: B, BMgBI; K, KpnI; P, PvuII; R, EcoRI; S, SalI. Solid black lines represent Southern blotting probes A and B used to screen for homologous recombination. Arrows show PCR primers (P1, P2, P3) used for genotyping the animals. ( b ) Southern blot analysis of genomic DNA extracted from wild-type (WT) and cyclin C +/F(Neo) (KI) ES cell clones. DNA was digested with EcoRI and hybridized with probe A (5’ end screening) or probe B (3’ end screening). The sizes of WT and recombinant alleles are shown. ( c ) PCR analysis of cyclin C F/F , C Δ/Δ and C +/Δ mice. The sizes of PCR products from the wild-type (C + ), “floxed” (C F ) and deleted (C Δ ) alleles are marked. ( d ) Left panel: the proportion of observed live cyclin C Δ/Δ embryos among all embryos at the indicated days of embryonic development (E8.5-10.5). Numbers in brackets denote dead embryos. The expected Mendelian proportion of cyclin C Δ/Δ embryos is also presented. Right panel: the photograph shows microscopic images of wild-type (WT) and cyclin C Δ/Δ (C-KO) littermates at E9.5. Scale bar, 0.5 mm. The absence of full-length cyclin C transcript in cyclin C Δ/Δ embryo was verified by RT-PCR. ( e ) Microscopic appearance of placentas from wild-type and cyclin C Δ/Δ (C-KO) embryos at E9.5. Histologic sections were stained with hematoxylin and eosin. Note underdeveloped labirynth layer (LB) in cyclin C-KO placenta. Scale bars, 50 μm.
    Figure Legend Snippet: Generation and analyses of cyclin C knockout mice. ( a ) Cyclin C gene targeting strategy. Coding exons are shown as filled boxes. Neo, neomycin phosphotransferase gene; loxP and FRT sequences are indicated as light blue triangles and dark blue rectangles, respectively. Restriction enzymes recognition sites: B, BMgBI; K, KpnI; P, PvuII; R, EcoRI; S, SalI. Solid black lines represent Southern blotting probes A and B used to screen for homologous recombination. Arrows show PCR primers (P1, P2, P3) used for genotyping the animals. ( b ) Southern blot analysis of genomic DNA extracted from wild-type (WT) and cyclin C +/F(Neo) (KI) ES cell clones. DNA was digested with EcoRI and hybridized with probe A (5’ end screening) or probe B (3’ end screening). The sizes of WT and recombinant alleles are shown. ( c ) PCR analysis of cyclin C F/F , C Δ/Δ and C +/Δ mice. The sizes of PCR products from the wild-type (C + ), “floxed” (C F ) and deleted (C Δ ) alleles are marked. ( d ) Left panel: the proportion of observed live cyclin C Δ/Δ embryos among all embryos at the indicated days of embryonic development (E8.5-10.5). Numbers in brackets denote dead embryos. The expected Mendelian proportion of cyclin C Δ/Δ embryos is also presented. Right panel: the photograph shows microscopic images of wild-type (WT) and cyclin C Δ/Δ (C-KO) littermates at E9.5. Scale bar, 0.5 mm. The absence of full-length cyclin C transcript in cyclin C Δ/Δ embryo was verified by RT-PCR. ( e ) Microscopic appearance of placentas from wild-type and cyclin C Δ/Δ (C-KO) embryos at E9.5. Histologic sections were stained with hematoxylin and eosin. Note underdeveloped labirynth layer (LB) in cyclin C-KO placenta. Scale bars, 50 μm.

    Techniques Used: Knock-Out, Mouse Assay, Southern Blot, Homologous Recombination, Polymerase Chain Reaction, Clone Assay, Recombinant, Reverse Transcription Polymerase Chain Reaction, Staining

    19) Product Images from "TAp73-induced phosphofructokinase-1 transcription promotes the Warburg effect and enhances cell proliferation"

    Article Title: TAp73-induced phosphofructokinase-1 transcription promotes the Warburg effect and enhances cell proliferation

    Journal: Nature Communications

    doi: 10.1038/s41467-018-07127-8

    PFKL is a physiologically relevant target of TAp73. a Schematic representation of human PFKL genomic structure. The sequences of potential p73 response elements RE1–3 and the corresponding mutant RE3 are shown. b , c Luciferase constructs containing RE1, RE2, and RE3 ( b ), or RE3 and mutant RE3 ( c ) were transfected into 293T cells together with Flag-TAp73α or vector control. Renilla vector pRL-CMV was used as a transfection internal control. The relative luciferase activity was normalized to the co-transfected Renila activity. Data are means ± S.D. ( n = 3). d Luciferase reporter constructs containing RE1, RE2, RE3, or RE3mut were transfected into 293T cells together vector control, p53, or TAp73. Renilla vector pRL-CMV was used as a transfection internal control. Relative levels of luciferase are shown. Data are means ± S.D. ( n = 3). Insert shows protein expression. e , f U2OS cells ( e ), or 293T cells transfected with control vector or Flag-TAp73 ( f ), were analyzed by ChIP assay using normal mouse IgG and anti-p73 antibody ( e ), or anti-Flag antibody ( f ). Bound DNA was amplified by PCR and quantified. Results are representative of three independent experiments. g , h U2OS ( g ) and HCT116 ( h ) cells transfected with the indicated siRNAs were analyzed for protein and mRNA expression. Results are representative of three independent experiments. i , j U2OS cells ( i ), or 293T cells transfected with Flag-p53 or vector control ( j ), were analyzed by ChIP assay using normal mouse IgG and anti-p53 antibody ( i ), or anti-Flag antibody ( j ). Bound DNA was amplified by PCR and quantified. Results are representative of three independent experiments. k p53 −/− HCT116 cells stable expressing Tet-inducible p53 were cultured in medium containing [1,2– 13 C 2 ]glucose and treated with doxycycline to induce p53 expression (Tet-on). p53, p73 and PFKL expressions were determined by Western blot analysis. TIGAR expression was analyzed by qRT-PCR. (means ± S.D., n = 3). Relative glycolytic flux is shown in Supplementary Fig. 4b
    Figure Legend Snippet: PFKL is a physiologically relevant target of TAp73. a Schematic representation of human PFKL genomic structure. The sequences of potential p73 response elements RE1–3 and the corresponding mutant RE3 are shown. b , c Luciferase constructs containing RE1, RE2, and RE3 ( b ), or RE3 and mutant RE3 ( c ) were transfected into 293T cells together with Flag-TAp73α or vector control. Renilla vector pRL-CMV was used as a transfection internal control. The relative luciferase activity was normalized to the co-transfected Renila activity. Data are means ± S.D. ( n = 3). d Luciferase reporter constructs containing RE1, RE2, RE3, or RE3mut were transfected into 293T cells together vector control, p53, or TAp73. Renilla vector pRL-CMV was used as a transfection internal control. Relative levels of luciferase are shown. Data are means ± S.D. ( n = 3). Insert shows protein expression. e , f U2OS cells ( e ), or 293T cells transfected with control vector or Flag-TAp73 ( f ), were analyzed by ChIP assay using normal mouse IgG and anti-p73 antibody ( e ), or anti-Flag antibody ( f ). Bound DNA was amplified by PCR and quantified. Results are representative of three independent experiments. g , h U2OS ( g ) and HCT116 ( h ) cells transfected with the indicated siRNAs were analyzed for protein and mRNA expression. Results are representative of three independent experiments. i , j U2OS cells ( i ), or 293T cells transfected with Flag-p53 or vector control ( j ), were analyzed by ChIP assay using normal mouse IgG and anti-p53 antibody ( i ), or anti-Flag antibody ( j ). Bound DNA was amplified by PCR and quantified. Results are representative of three independent experiments. k p53 −/− HCT116 cells stable expressing Tet-inducible p53 were cultured in medium containing [1,2– 13 C 2 ]glucose and treated with doxycycline to induce p53 expression (Tet-on). p53, p73 and PFKL expressions were determined by Western blot analysis. TIGAR expression was analyzed by qRT-PCR. (means ± S.D., n = 3). Relative glycolytic flux is shown in Supplementary Fig. 4b

    Techniques Used: Mutagenesis, Luciferase, Construct, Transfection, Plasmid Preparation, Activity Assay, Expressing, Chromatin Immunoprecipitation, Amplification, Polymerase Chain Reaction, Cell Culture, Western Blot, Quantitative RT-PCR

    20) Product Images from "hSSB1 associates with and promotes stability of the BLM helicase"

    Article Title: hSSB1 associates with and promotes stability of the BLM helicase

    Journal: BMC Molecular Biology

    doi: 10.1186/s12867-017-0090-3

    hSSB1 associates with BLM in cells. BLM ( a ) or hSSB1 ( b )-associating proteins were immunoprecipitated from U2OS whole cell lysates prepared from cells that had been either left untreated or exposed to 6 Gy ionising radiation (IR) and harvested after the indicated time periods. Control immunoprecipitations with an isotype IgG were performed from combination ( c ) samples comprised of equal amounts of each sample. Eluted proteins and whole cell lysates were separated by electrophoresis and immunoblotted with antibodies against BLM, hSSB1 and INTS3 ( b only). hSSB1 ( a ) or BLM ( b ) levels were determined by densitometry, normalised to the levels of BLM ( a ) or hSSB1 ( b ) as well as to the level of input protein and expressed relative to the untreated lane. c Line graph illustrating hSSB1: BLM association from three independent repeats of a and b . d BLM was immunoprecipitated from whole cell lysates, prepared from U2OS cells that were either untreated or had been treated with 2 mM hydroxyurea (HU) for 6 h. Eluted proteins and whole cell lysate samples (input) were immunoblotted with antibodies against BLM and hSSB1. hSSB1 levels were determined and expressed as per ( a ). e U2OS cells were transfected with plasmids encoding wild type (WT) or F98A 3× FLAG hSSB1, 24 h prior to cell lysis and immunoprecipitation of BLM-association proteins. Eluent was immunoblotted with antibodies against BLM, FLAG, INTS3, MRE11, RPA70 and RPA32. FLAG levels were determined by densitometry and normalised to the levels of BLM
    Figure Legend Snippet: hSSB1 associates with BLM in cells. BLM ( a ) or hSSB1 ( b )-associating proteins were immunoprecipitated from U2OS whole cell lysates prepared from cells that had been either left untreated or exposed to 6 Gy ionising radiation (IR) and harvested after the indicated time periods. Control immunoprecipitations with an isotype IgG were performed from combination ( c ) samples comprised of equal amounts of each sample. Eluted proteins and whole cell lysates were separated by electrophoresis and immunoblotted with antibodies against BLM, hSSB1 and INTS3 ( b only). hSSB1 ( a ) or BLM ( b ) levels were determined by densitometry, normalised to the levels of BLM ( a ) or hSSB1 ( b ) as well as to the level of input protein and expressed relative to the untreated lane. c Line graph illustrating hSSB1: BLM association from three independent repeats of a and b . d BLM was immunoprecipitated from whole cell lysates, prepared from U2OS cells that were either untreated or had been treated with 2 mM hydroxyurea (HU) for 6 h. Eluted proteins and whole cell lysate samples (input) were immunoblotted with antibodies against BLM and hSSB1. hSSB1 levels were determined and expressed as per ( a ). e U2OS cells were transfected with plasmids encoding wild type (WT) or F98A 3× FLAG hSSB1, 24 h prior to cell lysis and immunoprecipitation of BLM-association proteins. Eluent was immunoblotted with antibodies against BLM, FLAG, INTS3, MRE11, RPA70 and RPA32. FLAG levels were determined by densitometry and normalised to the levels of BLM

    Techniques Used: Immunoprecipitation, Electrophoresis, Transfection, Lysis

    21) Product Images from "Pumilio directs deadenylation-associated translational repression of the cyclin-dependent kinase 1 activator RGC-32"

    Article Title: Pumilio directs deadenylation-associated translational repression of the cyclin-dependent kinase 1 activator RGC-32

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky038

    The effect of Pumilio depletion on endogenous RGC-32 expression. ( A ) Silencing of Pumilio 1 and Pumilio 2 expression in the EBV-positive GM12878 LCL (latency III) using 500 nM of non targeting control siRNA or 200 or 500 nM of a mix of Pumilio 1 and Pumilio 2 siRNAs. Cells were harvested 48 h post transfection and analysed by western blotting for Pumilio 1, Pumilio 2, RGC-32 and actin (loading control). ( B ) Quantification of the Western blot results of two independent depletion experiments in GM12878 cells. Pumilio and RGC-32 signals were normalised to the actin control and then expressed relative to the level of expression in the cells transfected with the scrambled siRNA control. Data show the mean ± standard deviation of two independent depletion experiments. ( C ) Silencing of Pumilio 1 and Pumilio 2 expression in the EBV-positive Elijah BL line (latency I) using a mix of siRNAs. Cells were transfected with siRNAs, incubated for 24 h and then re-transfected with more siRNAs and harvested after a further 24 h to achieve optimal depletion. Pumilio 1, Pumilio 2 and actin levels were determined by western blotting. Samples were re-analysed for RGC-32 expression alongside a positive control for RGC-32 expression (LCL). ( D ) Quantification of the western blot results of two independent depletion experiments in Elijah cells as in (B).
    Figure Legend Snippet: The effect of Pumilio depletion on endogenous RGC-32 expression. ( A ) Silencing of Pumilio 1 and Pumilio 2 expression in the EBV-positive GM12878 LCL (latency III) using 500 nM of non targeting control siRNA or 200 or 500 nM of a mix of Pumilio 1 and Pumilio 2 siRNAs. Cells were harvested 48 h post transfection and analysed by western blotting for Pumilio 1, Pumilio 2, RGC-32 and actin (loading control). ( B ) Quantification of the Western blot results of two independent depletion experiments in GM12878 cells. Pumilio and RGC-32 signals were normalised to the actin control and then expressed relative to the level of expression in the cells transfected with the scrambled siRNA control. Data show the mean ± standard deviation of two independent depletion experiments. ( C ) Silencing of Pumilio 1 and Pumilio 2 expression in the EBV-positive Elijah BL line (latency I) using a mix of siRNAs. Cells were transfected with siRNAs, incubated for 24 h and then re-transfected with more siRNAs and harvested after a further 24 h to achieve optimal depletion. Pumilio 1, Pumilio 2 and actin levels were determined by western blotting. Samples were re-analysed for RGC-32 expression alongside a positive control for RGC-32 expression (LCL). ( D ) Quantification of the western blot results of two independent depletion experiments in Elijah cells as in (B).

    Techniques Used: Expressing, Transfection, Western Blot, Standard Deviation, Incubation, Positive Control

    Analysis of Pumilio expression and Pumilio binding in B cell lines. ( A ) Western blot analysis of Pumilio 1 and Pumilio 2 protein expression in EBV negative (–ve), EBV-infected latency I (I) and latency III (III) cell lines. Actin levels serve as a loading control. ( B ) RNA immunoprecipitation analysis using anti-Pumilio 1 antibodies in cross-linked Mutu I (latency 1) and Mutu III (latency III) cell lines. Precipitated RNA was reverse transcribed and analysed by QPCR against a standard curve of RNA extracted from an input sample from each cell line. Percentage input values for each Pumilio 1 immunopreciptation were divided by signals obtained in the IgG control immunoprecipitation and then normalised to background enrichment for a non-target mRNA (GAPDH). Cyclin B is a known target of Pumilio and was used as a positive control for mRNA binding. Data show the mean ± standard deviation of two independent experiments. ( C ) RNA immunoprecipitation for Pumilio 1 carried out in Akata (latency I) and GM12878 (latency III) cells as in (B).
    Figure Legend Snippet: Analysis of Pumilio expression and Pumilio binding in B cell lines. ( A ) Western blot analysis of Pumilio 1 and Pumilio 2 protein expression in EBV negative (–ve), EBV-infected latency I (I) and latency III (III) cell lines. Actin levels serve as a loading control. ( B ) RNA immunoprecipitation analysis using anti-Pumilio 1 antibodies in cross-linked Mutu I (latency 1) and Mutu III (latency III) cell lines. Precipitated RNA was reverse transcribed and analysed by QPCR against a standard curve of RNA extracted from an input sample from each cell line. Percentage input values for each Pumilio 1 immunopreciptation were divided by signals obtained in the IgG control immunoprecipitation and then normalised to background enrichment for a non-target mRNA (GAPDH). Cyclin B is a known target of Pumilio and was used as a positive control for mRNA binding. Data show the mean ± standard deviation of two independent experiments. ( C ) RNA immunoprecipitation for Pumilio 1 carried out in Akata (latency I) and GM12878 (latency III) cells as in (B).

    Techniques Used: Expressing, Binding Assay, Western Blot, Infection, Immunoprecipitation, Real-time Polymerase Chain Reaction, Positive Control, Standard Deviation

    The effects of Pumilio depletion on polyadenylation of the endogenous RGC-32 mRNA. ( A ) ePAT analysis of the length of the endogenous RGC-32 mRNA polyA tail in unstransfected (Unt) GM12878 latency III cells, and in cells transfected with non targeting control siRNA (500 nM) or 200 nM of a 1:1 mix of Pumilio 1 and Pumilio 2 siRNAs. ePAT analysis of the polyA tail of the endogenous GAPDH mRNA was also measured as a control. ( B ) ImageJ quantitation of the agarose gels shown in (A). The areas boxed by dotted lines show the size of the most abundant polyadenylated mRNA species. The arrow shows the RGC-32 mRNA species with a longer polyA tail detected in cells transfected with Pumilio targeting siRNAs.
    Figure Legend Snippet: The effects of Pumilio depletion on polyadenylation of the endogenous RGC-32 mRNA. ( A ) ePAT analysis of the length of the endogenous RGC-32 mRNA polyA tail in unstransfected (Unt) GM12878 latency III cells, and in cells transfected with non targeting control siRNA (500 nM) or 200 nM of a 1:1 mix of Pumilio 1 and Pumilio 2 siRNAs. ePAT analysis of the polyA tail of the endogenous GAPDH mRNA was also measured as a control. ( B ) ImageJ quantitation of the agarose gels shown in (A). The areas boxed by dotted lines show the size of the most abundant polyadenylated mRNA species. The arrow shows the RGC-32 mRNA species with a longer polyA tail detected in cells transfected with Pumilio targeting siRNAs.

    Techniques Used: Transfection, Quantitation Assay

    22) Product Images from "Pumilio directs deadenylation-associated translational repression of the cyclin-dependent kinase 1 activator RGC-32"

    Article Title: Pumilio directs deadenylation-associated translational repression of the cyclin-dependent kinase 1 activator RGC-32

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky038

    The effect of Pumilio depletion on endogenous RGC-32 expression. ( A ) Silencing of Pumilio 1 and Pumilio 2 expression in the EBV-positive GM12878 LCL (latency III) using 500 nM of non targeting control siRNA or 200 or 500 nM of a mix of Pumilio 1 and Pumilio 2 siRNAs. Cells were harvested 48 h post transfection and analysed by western blotting for Pumilio 1, Pumilio 2, RGC-32 and actin (loading control). ( B ) Quantification of the Western blot results of two independent depletion experiments in GM12878 cells. Pumilio and RGC-32 signals were normalised to the actin control and then expressed relative to the level of expression in the cells transfected with the scrambled siRNA control. Data show the mean ± standard deviation of two independent depletion experiments. ( C ) Silencing of Pumilio 1 and Pumilio 2 expression in the EBV-positive Elijah BL line (latency I) using a mix of siRNAs. Cells were transfected with siRNAs, incubated for 24 h and then re-transfected with more siRNAs and harvested after a further 24 h to achieve optimal depletion. Pumilio 1, Pumilio 2 and actin levels were determined by western blotting. Samples were re-analysed for RGC-32 expression alongside a positive control for RGC-32 expression (LCL). ( D ) Quantification of the western blot results of two independent depletion experiments in Elijah cells as in (B).
    Figure Legend Snippet: The effect of Pumilio depletion on endogenous RGC-32 expression. ( A ) Silencing of Pumilio 1 and Pumilio 2 expression in the EBV-positive GM12878 LCL (latency III) using 500 nM of non targeting control siRNA or 200 or 500 nM of a mix of Pumilio 1 and Pumilio 2 siRNAs. Cells were harvested 48 h post transfection and analysed by western blotting for Pumilio 1, Pumilio 2, RGC-32 and actin (loading control). ( B ) Quantification of the Western blot results of two independent depletion experiments in GM12878 cells. Pumilio and RGC-32 signals were normalised to the actin control and then expressed relative to the level of expression in the cells transfected with the scrambled siRNA control. Data show the mean ± standard deviation of two independent depletion experiments. ( C ) Silencing of Pumilio 1 and Pumilio 2 expression in the EBV-positive Elijah BL line (latency I) using a mix of siRNAs. Cells were transfected with siRNAs, incubated for 24 h and then re-transfected with more siRNAs and harvested after a further 24 h to achieve optimal depletion. Pumilio 1, Pumilio 2 and actin levels were determined by western blotting. Samples were re-analysed for RGC-32 expression alongside a positive control for RGC-32 expression (LCL). ( D ) Quantification of the western blot results of two independent depletion experiments in Elijah cells as in (B).

    Techniques Used: Expressing, Transfection, Western Blot, Standard Deviation, Incubation, Positive Control

    Analysis of Pumilio expression and Pumilio binding in B cell lines. ( A ) Western blot analysis of Pumilio 1 and Pumilio 2 protein expression in EBV negative (–ve), EBV-infected latency I (I) and latency III (III) cell lines. Actin levels serve as a loading control. ( B ) RNA immunoprecipitation analysis using anti-Pumilio 1 antibodies in cross-linked Mutu I (latency 1) and Mutu III (latency III) cell lines. Precipitated RNA was reverse transcribed and analysed by QPCR against a standard curve of RNA extracted from an input sample from each cell line. Percentage input values for each Pumilio 1 immunopreciptation were divided by signals obtained in the IgG control immunoprecipitation and then normalised to background enrichment for a non-target mRNA (GAPDH). Cyclin B is a known target of Pumilio and was used as a positive control for mRNA binding. Data show the mean ± standard deviation of two independent experiments. ( C ) RNA immunoprecipitation for Pumilio 1 carried out in Akata (latency I) and GM12878 (latency III) cells as in (B).
    Figure Legend Snippet: Analysis of Pumilio expression and Pumilio binding in B cell lines. ( A ) Western blot analysis of Pumilio 1 and Pumilio 2 protein expression in EBV negative (–ve), EBV-infected latency I (I) and latency III (III) cell lines. Actin levels serve as a loading control. ( B ) RNA immunoprecipitation analysis using anti-Pumilio 1 antibodies in cross-linked Mutu I (latency 1) and Mutu III (latency III) cell lines. Precipitated RNA was reverse transcribed and analysed by QPCR against a standard curve of RNA extracted from an input sample from each cell line. Percentage input values for each Pumilio 1 immunopreciptation were divided by signals obtained in the IgG control immunoprecipitation and then normalised to background enrichment for a non-target mRNA (GAPDH). Cyclin B is a known target of Pumilio and was used as a positive control for mRNA binding. Data show the mean ± standard deviation of two independent experiments. ( C ) RNA immunoprecipitation for Pumilio 1 carried out in Akata (latency I) and GM12878 (latency III) cells as in (B).

    Techniques Used: Expressing, Binding Assay, Western Blot, Infection, Immunoprecipitation, Real-time Polymerase Chain Reaction, Positive Control, Standard Deviation

    The effects of Pumilio depletion on polyadenylation of the endogenous RGC-32 mRNA. ( A ) ePAT analysis of the length of the endogenous RGC-32 mRNA polyA tail in unstransfected (Unt) GM12878 latency III cells, and in cells transfected with non targeting control siRNA (500 nM) or 200 nM of a 1:1 mix of Pumilio 1 and Pumilio 2 siRNAs. ePAT analysis of the polyA tail of the endogenous GAPDH mRNA was also measured as a control. ( B ) ImageJ quantitation of the agarose gels shown in (A). The areas boxed by dotted lines show the size of the most abundant polyadenylated mRNA species. The arrow shows the RGC-32 mRNA species with a longer polyA tail detected in cells transfected with Pumilio targeting siRNAs.
    Figure Legend Snippet: The effects of Pumilio depletion on polyadenylation of the endogenous RGC-32 mRNA. ( A ) ePAT analysis of the length of the endogenous RGC-32 mRNA polyA tail in unstransfected (Unt) GM12878 latency III cells, and in cells transfected with non targeting control siRNA (500 nM) or 200 nM of a 1:1 mix of Pumilio 1 and Pumilio 2 siRNAs. ePAT analysis of the polyA tail of the endogenous GAPDH mRNA was also measured as a control. ( B ) ImageJ quantitation of the agarose gels shown in (A). The areas boxed by dotted lines show the size of the most abundant polyadenylated mRNA species. The arrow shows the RGC-32 mRNA species with a longer polyA tail detected in cells transfected with Pumilio targeting siRNAs.

    Techniques Used: Transfection, Quantitation Assay

    23) Product Images from "FUS/TLS contributes to replication-dependent histone gene expression by interaction with U7 snRNPs and histone-specific transcription factors"

    Article Title: FUS/TLS contributes to replication-dependent histone gene expression by interaction with U7 snRNPs and histone-specific transcription factors

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv794

    U7 snRNA can be enriched by FUS immunoprecipitation. HeLa nuclear extract was subjected to immunoprecipitation with two different anti-FUS antibodies (dark grey) and one anti-V5 antibody (light grey; negative control), and the levels of U7 and U6 snRNAs as well as those of 7SL RNA were quantitated by RT-qPCR. The amounts detected in the immunprecipitates (IP) are expressed as percent of the input. Error bars indicate standard deviations (SD) of three technical replicates for each antibody (i.e. six and three values for anti-FUS and anti-V5, respectively). P -values were calculated using Student's T-test, and statistical significance is represented as follows: ** P ≤ 0.01.
    Figure Legend Snippet: U7 snRNA can be enriched by FUS immunoprecipitation. HeLa nuclear extract was subjected to immunoprecipitation with two different anti-FUS antibodies (dark grey) and one anti-V5 antibody (light grey; negative control), and the levels of U7 and U6 snRNAs as well as those of 7SL RNA were quantitated by RT-qPCR. The amounts detected in the immunprecipitates (IP) are expressed as percent of the input. Error bars indicate standard deviations (SD) of three technical replicates for each antibody (i.e. six and three values for anti-FUS and anti-V5, respectively). P -values were calculated using Student's T-test, and statistical significance is represented as follows: ** P ≤ 0.01.

    Techniques Used: Immunoprecipitation, Negative Control, Quantitative RT-PCR

    24) Product Images from "hPaf1/PD2 interacts with OCT3/4 to promote self-renewal of ovarian cancer stem cells"

    Article Title: hPaf1/PD2 interacts with OCT3/4 to promote self-renewal of ovarian cancer stem cells

    Journal: Oncotarget

    doi: 10.18632/oncotarget.14775

    PAF Complex independent interaction of hPaf1/PD2 with OCT 3/4 in ovarian cancer stem cells ( A ) Co-immunoprecipitation assay showed that hPaf1/PD2 interacts with OCT3/4. hPaf1/PD2 antibody was used for pulldown and immunoprecipitates were probed with OCT3/4 antibody. Absence of non-specific binding was confirmed by including an IgG control. ( B ) We also performed reciprocal co-immunoprecipitation assay to pull down OCT3/4 and probed with hPaf1/PD2 antibody. We did not observe any interaction of OCT3/4 with other PAF complex components such as Leo1, Ctr9 and Parafibromin. Absence of non-specific binding was confirmed by including an IgG control. ( C ) Co-localization of hPaf1/PD2 with OCT3/4 was also observed using confocal microscopy. The box indicates a zoomed image depicting perinuclear and nuclear co-localization of hPaf1/PD2 with OCT3/4. ( D ) Western blotting analysis revealed that there was no change in expression of other PAF complex components such as Ctr9, Leo1, and Parafibromin on knockdown of hPaf1/PD2. Equal amount of protein was loaded in each well. β-actin was used as a loading control.
    Figure Legend Snippet: PAF Complex independent interaction of hPaf1/PD2 with OCT 3/4 in ovarian cancer stem cells ( A ) Co-immunoprecipitation assay showed that hPaf1/PD2 interacts with OCT3/4. hPaf1/PD2 antibody was used for pulldown and immunoprecipitates were probed with OCT3/4 antibody. Absence of non-specific binding was confirmed by including an IgG control. ( B ) We also performed reciprocal co-immunoprecipitation assay to pull down OCT3/4 and probed with hPaf1/PD2 antibody. We did not observe any interaction of OCT3/4 with other PAF complex components such as Leo1, Ctr9 and Parafibromin. Absence of non-specific binding was confirmed by including an IgG control. ( C ) Co-localization of hPaf1/PD2 with OCT3/4 was also observed using confocal microscopy. The box indicates a zoomed image depicting perinuclear and nuclear co-localization of hPaf1/PD2 with OCT3/4. ( D ) Western blotting analysis revealed that there was no change in expression of other PAF complex components such as Ctr9, Leo1, and Parafibromin on knockdown of hPaf1/PD2. Equal amount of protein was loaded in each well. β-actin was used as a loading control.

    Techniques Used: Co-Immunoprecipitation Assay, Binding Assay, Confocal Microscopy, Western Blot, Expressing

    25) Product Images from "RPA70 depletion induces hSSB1/2-INTS3 complex to initiate ATR signaling"

    Article Title: RPA70 depletion induces hSSB1/2-INTS3 complex to initiate ATR signaling

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv369

    ATR-ATRIP complex associates with hSSB1/2-INTS3. ( A and B ) 293T cells were co-transfected with plasmids expressing HA-ATR (1–400 aa), HA-ATRIP or a non-specific protein (HA-NS) in combination with Myc-INTS3 or Myc-hSSB1 as indicated followed by immunoprecipitation (IP) with HA antibody. The lysates were normalized for the expression of HA-tagged and Myc-tagged proteins, shown by immunoblotting (IB) in the first and second panels, respectively. The bottom panel displays the co-immunoprecipitated Myc-tagged protein. HA-ATR (hollow arrowhead), HA-ATRIP (black arrowhead) and HA-NS (double arrowhead) have been marked while (*) displays multiple expression products of the non-specific protein (HA-NS). The expression of an endogenous non-specific protein (LC) in different transfected samples as visualized by Ponceau S staining has been shown. Note that lanes 2 and 3 of the bottom panel in part B are separated by an intervening lane to prevent any spill over artifacts. ( C and D ) 293T cells were co-transfected with plasmids expressing HA-ATR (1–400 aa) or HA-ATRIP in combination with Myc-INTS3 or a non-specific protein (Myc-NS) as indicated followed by immunoprecipitation with Myc antibody. The expression of Myc-tagged and HA-tagged proteins have been shown in the first and second panels respectively while the bottom panel displays the co-immunoprecipitated HA-tagged protein (black arrowhead). Myc-INTS3 and Myc-NS have similar mobility. Note that lanes 1 and 2 of the bottom panel in part D are separated by an intervening lane to prevent any spill over artifacts and other details are same as parts A and B.
    Figure Legend Snippet: ATR-ATRIP complex associates with hSSB1/2-INTS3. ( A and B ) 293T cells were co-transfected with plasmids expressing HA-ATR (1–400 aa), HA-ATRIP or a non-specific protein (HA-NS) in combination with Myc-INTS3 or Myc-hSSB1 as indicated followed by immunoprecipitation (IP) with HA antibody. The lysates were normalized for the expression of HA-tagged and Myc-tagged proteins, shown by immunoblotting (IB) in the first and second panels, respectively. The bottom panel displays the co-immunoprecipitated Myc-tagged protein. HA-ATR (hollow arrowhead), HA-ATRIP (black arrowhead) and HA-NS (double arrowhead) have been marked while (*) displays multiple expression products of the non-specific protein (HA-NS). The expression of an endogenous non-specific protein (LC) in different transfected samples as visualized by Ponceau S staining has been shown. Note that lanes 2 and 3 of the bottom panel in part B are separated by an intervening lane to prevent any spill over artifacts. ( C and D ) 293T cells were co-transfected with plasmids expressing HA-ATR (1–400 aa) or HA-ATRIP in combination with Myc-INTS3 or a non-specific protein (Myc-NS) as indicated followed by immunoprecipitation with Myc antibody. The expression of Myc-tagged and HA-tagged proteins have been shown in the first and second panels respectively while the bottom panel displays the co-immunoprecipitated HA-tagged protein (black arrowhead). Myc-INTS3 and Myc-NS have similar mobility. Note that lanes 1 and 2 of the bottom panel in part D are separated by an intervening lane to prevent any spill over artifacts and other details are same as parts A and B.

    Techniques Used: Transfection, Expressing, Immunoprecipitation, Staining

    Chk1 phosphorylation in the absence of RPA70 is dependent on single-strand binding protein complex, hSSB1/2-INTS3. ( A ) HeLa cells were transfected on three consecutive days with GL2 or RPA70 siRNA in combination with INTS3 siRNA as indicated and the levels of RPA70, INTS3, total and phosphorylated-Chk1 were assayed. ( B ) HeLa cells were transfected on three consecutive days with GL2 or RPA70 siRNA in combination with HSSB1 and/or HSSB2 siRNAs as indicated and the levels of RPA70, hSSB1, hSSB2, total and phosphorylated-Chk1 were assayed. * points to a cross-reactive band while LC refers to the protein loading control. The numbers in parts A and B indicate phosphorylated-Chk1 levels following RPA70 depletion alone or in combination with INTS3 or hSSB1 2 after normalization with the protein loading control. ( C and D ) Different siRNA duplexes confirm the requirement of the hSSB1/2-INTS3 complex for Chk1 phosphorylation in RPA-depleted cells. HeLa cells were transfected on three consecutive days with GL2 or RPA70 siRNA in combination with different siRNA duplexes ( INTS3(2), HSSB2 HSSB1(2) ) than used in parts A and B. The numbers in parts C and D indicate phosphorylated-Chk1 levels following RPA70 depletion alone or in combination with INTS3 or hSSB1 2 after normalization with the protein loading control.
    Figure Legend Snippet: Chk1 phosphorylation in the absence of RPA70 is dependent on single-strand binding protein complex, hSSB1/2-INTS3. ( A ) HeLa cells were transfected on three consecutive days with GL2 or RPA70 siRNA in combination with INTS3 siRNA as indicated and the levels of RPA70, INTS3, total and phosphorylated-Chk1 were assayed. ( B ) HeLa cells were transfected on three consecutive days with GL2 or RPA70 siRNA in combination with HSSB1 and/or HSSB2 siRNAs as indicated and the levels of RPA70, hSSB1, hSSB2, total and phosphorylated-Chk1 were assayed. * points to a cross-reactive band while LC refers to the protein loading control. The numbers in parts A and B indicate phosphorylated-Chk1 levels following RPA70 depletion alone or in combination with INTS3 or hSSB1 2 after normalization with the protein loading control. ( C and D ) Different siRNA duplexes confirm the requirement of the hSSB1/2-INTS3 complex for Chk1 phosphorylation in RPA-depleted cells. HeLa cells were transfected on three consecutive days with GL2 or RPA70 siRNA in combination with different siRNA duplexes ( INTS3(2), HSSB2 HSSB1(2) ) than used in parts A and B. The numbers in parts C and D indicate phosphorylated-Chk1 levels following RPA70 depletion alone or in combination with INTS3 or hSSB1 2 after normalization with the protein loading control.

    Techniques Used: Binding Assay, Transfection, Recombinase Polymerase Amplification

    INTS3 and hSSB1 form sub-nuclear foci after RP70 depletion. ( A and B ) HeLa cells transfected on three consecutive days with GL2 or RPA70 siRNA in combination with HSSB1 or INTS3 siRNA as indicated were visualized for hSSB1 and INTS3 foci by immunofluorescence with rabbit anti-hSSB1 and goat anti-INTS3 antibodies respectively. Right panel displays the DAPI staining for each sample. Co-depletion of hSSB1 (A) or INTS3 (B) along with RPA70 confirms that the immunofluorescence signal is from the respective proteins. ( C ) Quantification of INTS3 and hSSB1 foci observed in the experiments described in parts A and B. Cells from GL2 or RPA70 siRNA transfected samples were scored for INTS3 and hSSB1 foci and are expressed as a percentage of total cells from each group. Data are represented as the mean ± SE. P- values were calculated using two-tailed t -test which displays that RPA70 siRNA transfected samples are significantly different from control GL2 siRNA transfected samples (* P- value
    Figure Legend Snippet: INTS3 and hSSB1 form sub-nuclear foci after RP70 depletion. ( A and B ) HeLa cells transfected on three consecutive days with GL2 or RPA70 siRNA in combination with HSSB1 or INTS3 siRNA as indicated were visualized for hSSB1 and INTS3 foci by immunofluorescence with rabbit anti-hSSB1 and goat anti-INTS3 antibodies respectively. Right panel displays the DAPI staining for each sample. Co-depletion of hSSB1 (A) or INTS3 (B) along with RPA70 confirms that the immunofluorescence signal is from the respective proteins. ( C ) Quantification of INTS3 and hSSB1 foci observed in the experiments described in parts A and B. Cells from GL2 or RPA70 siRNA transfected samples were scored for INTS3 and hSSB1 foci and are expressed as a percentage of total cells from each group. Data are represented as the mean ± SE. P- values were calculated using two-tailed t -test which displays that RPA70 siRNA transfected samples are significantly different from control GL2 siRNA transfected samples (* P- value

    Techniques Used: Transfection, Immunofluorescence, Staining, Two Tailed Test

    Inactivation of hSSB1/2-INTS3 complex along with RPA debilitates the ability of cells to phosphorylate Chk1. ( A ) HeLa cells were transfected on three consecutive days with GL2 or RPA70 siRNA in combination with INTS3 siRNA, as indicated. 24 h after the last transfection, the cells were either left non-irradiated (top four panels) or UV-irradiated (bottom four panels) as indicated, followed by co-immunofluorescence with mouse anti-RPA70 and rabbit anti-phospho-Chk1 (Ser345) antibodies. The scale bar is 10 μm. Cells were scored for RPA70 and P-Chk1 signals and the percentage of total cells from each group displaying RPA70 depletion and phosphorylated-Chk1 have been indicated. All data represent the mean ± SE of two independent experiments. * indicates that the siRNA depletion was carried out in a separate experiment wherein 38.45% of UV-irradiated GL2 cells displayed phosphorylated-Chk1. # P- values were calculated using two-tailed t -test which displays that the Chk1 phosphorylation observed in the non-UV-irradiated RPA70 siRNA transfected sample is significantly different from RPA70+INTS3 siRNA transfected sample ( # P- value = 0.026). ## The P- value of UV-irradiated RPA70 siRNA transfected sample versus RPA70+INTS3 siRNA transfected sample is 0.033. ( B ) A model for ATR-ATRIP recruitment by hSSB1/2-INTS3 complex. Single-stranded DNA (ssDNA) generated at the sites of genomic stress is coated by hSSB1/2-INTS3 complex in the absence of RPA. The N-terminus of INTS3 associates with the oligonucleotide/oligosaccharide-binding fold of hSSB1/2, which binds to the ssDNA ( 23 , 25 – 27 ). ATR-ATRIP complex is then recruited to the hSSB1/2-INTS3 bound ssDNA. Rad9-Hus1-Rad1 clamp (9–1–1 complex) is recruited to ssDNA independently of ATRIP but it is not clear which single-stranded DNA-binding protein (SSB?) associates with it in the absence of RPA. TopBP1 binds to Rad17 loaded Rad9 to come in close proximity to activate ATR, which phosphorylates Chk1. It is likely that many yet unidentified factors participate in RPA-independent ATR activation.
    Figure Legend Snippet: Inactivation of hSSB1/2-INTS3 complex along with RPA debilitates the ability of cells to phosphorylate Chk1. ( A ) HeLa cells were transfected on three consecutive days with GL2 or RPA70 siRNA in combination with INTS3 siRNA, as indicated. 24 h after the last transfection, the cells were either left non-irradiated (top four panels) or UV-irradiated (bottom four panels) as indicated, followed by co-immunofluorescence with mouse anti-RPA70 and rabbit anti-phospho-Chk1 (Ser345) antibodies. The scale bar is 10 μm. Cells were scored for RPA70 and P-Chk1 signals and the percentage of total cells from each group displaying RPA70 depletion and phosphorylated-Chk1 have been indicated. All data represent the mean ± SE of two independent experiments. * indicates that the siRNA depletion was carried out in a separate experiment wherein 38.45% of UV-irradiated GL2 cells displayed phosphorylated-Chk1. # P- values were calculated using two-tailed t -test which displays that the Chk1 phosphorylation observed in the non-UV-irradiated RPA70 siRNA transfected sample is significantly different from RPA70+INTS3 siRNA transfected sample ( # P- value = 0.026). ## The P- value of UV-irradiated RPA70 siRNA transfected sample versus RPA70+INTS3 siRNA transfected sample is 0.033. ( B ) A model for ATR-ATRIP recruitment by hSSB1/2-INTS3 complex. Single-stranded DNA (ssDNA) generated at the sites of genomic stress is coated by hSSB1/2-INTS3 complex in the absence of RPA. The N-terminus of INTS3 associates with the oligonucleotide/oligosaccharide-binding fold of hSSB1/2, which binds to the ssDNA ( 23 , 25 – 27 ). ATR-ATRIP complex is then recruited to the hSSB1/2-INTS3 bound ssDNA. Rad9-Hus1-Rad1 clamp (9–1–1 complex) is recruited to ssDNA independently of ATRIP but it is not clear which single-stranded DNA-binding protein (SSB?) associates with it in the absence of RPA. TopBP1 binds to Rad17 loaded Rad9 to come in close proximity to activate ATR, which phosphorylates Chk1. It is likely that many yet unidentified factors participate in RPA-independent ATR activation.

    Techniques Used: Recombinase Polymerase Amplification, Transfection, Irradiation, Immunofluorescence, Two Tailed Test, Generated, Binding Assay, Activation Assay

    The single-strand binding protein complex, hSSB1/2-INTS3 recruits ATRIP to single-stranded DNA. ( A ) Myc-hSSB1, Myc-INTS3, HA-ATRIP and HA-tagged non-specific control protein (HA-NS) were individually expressed in 293T cells, purified by immunoprecipitation with anti-Myc or anti-HA antibodies and were eluted with Myc or HA peptides. For the single-stranded DNA-binding assay, streptavidin-agarose was incubated with non-biotinylated (lane 2) or biotinylated (lane 3) single-stranded DNA followed by incubation with Myc-hSSB1 and Myc-INTS3. Next, HA-ATRIP or HA-NS purified from 293T cells were incubated with streptavidin-agarose bound biotinylated ssDNA either in the absence (lanes 4 and 6) or presence (lanes 5 and 7) of bound Myc-hSSB1 and Myc-INTS3. After washing, the bound proteins were identified by immunoblotting with anti-HA (top panel) and anti-Myc (bottom panel) antibodies. 10% of NS and ATRIP utilized for binding to streptavidin-agarose has been shown in lanes 1 and 8 respectively and specific proteins have been marked by arrowheads. The control protein (NS) did not bind to hSSB1-INTS3 complex, ruling out non-specific association. The numbers indicate relative binding of HA-ATRIP to ssDNA in the absence or presence of Myc-hSSB1 and Myc-INTS3. * P -value was calculated using two-tailed t -test which displays that the ATRIP binding observed in the absence or presence of hSSB1-INTS3 complex is significantly different (* P- value = 0.043). ( B ) RPA complex is absent in Myc-hSSB1 and Myc-INTS3 immunoprecipitates. Myc-hSSB1, Myc-INTS3, HA-ATRIP and HA-NS proteins expressed in 293T cells and purified by elution with Myc or HA peptides following immunoprecipitation were immunoblotted with anti-RPA70 (top panel) and anti-RPA32 (second panel) antibodies for detecting endogenous RPA70 and RPA32. As reported earlier, RPA complex physically associates with ATRIP but is absent from hSSB1-INTS3 complex. Note the high sensitivity of detection of endogenous RPA70 and RPA32 in 293T cell lysate. HA-ATRIP (hollow arrowhead), HA-NS (black arrow) Myc-INTS3 (black arrowhead) and Myc-hSSB1 (shaded arrowhead) have been marked.
    Figure Legend Snippet: The single-strand binding protein complex, hSSB1/2-INTS3 recruits ATRIP to single-stranded DNA. ( A ) Myc-hSSB1, Myc-INTS3, HA-ATRIP and HA-tagged non-specific control protein (HA-NS) were individually expressed in 293T cells, purified by immunoprecipitation with anti-Myc or anti-HA antibodies and were eluted with Myc or HA peptides. For the single-stranded DNA-binding assay, streptavidin-agarose was incubated with non-biotinylated (lane 2) or biotinylated (lane 3) single-stranded DNA followed by incubation with Myc-hSSB1 and Myc-INTS3. Next, HA-ATRIP or HA-NS purified from 293T cells were incubated with streptavidin-agarose bound biotinylated ssDNA either in the absence (lanes 4 and 6) or presence (lanes 5 and 7) of bound Myc-hSSB1 and Myc-INTS3. After washing, the bound proteins were identified by immunoblotting with anti-HA (top panel) and anti-Myc (bottom panel) antibodies. 10% of NS and ATRIP utilized for binding to streptavidin-agarose has been shown in lanes 1 and 8 respectively and specific proteins have been marked by arrowheads. The control protein (NS) did not bind to hSSB1-INTS3 complex, ruling out non-specific association. The numbers indicate relative binding of HA-ATRIP to ssDNA in the absence or presence of Myc-hSSB1 and Myc-INTS3. * P -value was calculated using two-tailed t -test which displays that the ATRIP binding observed in the absence or presence of hSSB1-INTS3 complex is significantly different (* P- value = 0.043). ( B ) RPA complex is absent in Myc-hSSB1 and Myc-INTS3 immunoprecipitates. Myc-hSSB1, Myc-INTS3, HA-ATRIP and HA-NS proteins expressed in 293T cells and purified by elution with Myc or HA peptides following immunoprecipitation were immunoblotted with anti-RPA70 (top panel) and anti-RPA32 (second panel) antibodies for detecting endogenous RPA70 and RPA32. As reported earlier, RPA complex physically associates with ATRIP but is absent from hSSB1-INTS3 complex. Note the high sensitivity of detection of endogenous RPA70 and RPA32 in 293T cell lysate. HA-ATRIP (hollow arrowhead), HA-NS (black arrow) Myc-INTS3 (black arrowhead) and Myc-hSSB1 (shaded arrowhead) have been marked.

    Techniques Used: Binding Assay, Purification, Immunoprecipitation, DNA Binding Assay, Incubation, Two Tailed Test, Recombinase Polymerase Amplification

    26) Product Images from "Aurora-A controls pre-replicative complex assembly and DNA replication by stabilizing geminin in mitosis"

    Article Title: Aurora-A controls pre-replicative complex assembly and DNA replication by stabilizing geminin in mitosis

    Journal: Nature Communications

    doi: 10.1038/ncomms2859

    Downregulation of Cdt1 by geminin or Aurora-A mitotic depletion is caused by SCF Skp2 . ( a ) U2OS cells were treated with geminin or Aurora-A siRNA during release from the second thymidine block and collected immediately after release from the Noc block as described in Fig. 4a . Cells were treated with 25 μM MG132 for 5 h before mitotic shake-off. The soluble fraction was separated and immunoblotted for the indicated proteins. MW, molecular weight in kDa. ( b ) U2OS cells were treated with geminin or Aurora-A siRNA during release from the second thymidine block and collected immediately after release from the Noc block as described in Fig. 4a . Cells were treated with 1 μM MLN4924 for 2 h before mitotic shake-off. The soluble fraction was separated and immunoblotted for the indicated proteins. ( c ) U2OS cells were treated with geminin siRNA, with or without Skp2 or Cdt2 siRNA, during release from the second thymidine block and collected immediately after release from the Noc block as described in Fig. 4a . The soluble fraction was separated and immunoblotted for the indicated proteins. MW, molecular weight in kDa.
    Figure Legend Snippet: Downregulation of Cdt1 by geminin or Aurora-A mitotic depletion is caused by SCF Skp2 . ( a ) U2OS cells were treated with geminin or Aurora-A siRNA during release from the second thymidine block and collected immediately after release from the Noc block as described in Fig. 4a . Cells were treated with 25 μM MG132 for 5 h before mitotic shake-off. The soluble fraction was separated and immunoblotted for the indicated proteins. MW, molecular weight in kDa. ( b ) U2OS cells were treated with geminin or Aurora-A siRNA during release from the second thymidine block and collected immediately after release from the Noc block as described in Fig. 4a . Cells were treated with 1 μM MLN4924 for 2 h before mitotic shake-off. The soluble fraction was separated and immunoblotted for the indicated proteins. ( c ) U2OS cells were treated with geminin siRNA, with or without Skp2 or Cdt2 siRNA, during release from the second thymidine block and collected immediately after release from the Noc block as described in Fig. 4a . The soluble fraction was separated and immunoblotted for the indicated proteins. MW, molecular weight in kDa.

    Techniques Used: Blocking Assay, Molecular Weight

    27) Product Images from "An extracellular matrix-specific GEF-GAP interaction regulates Rho GTPase crosstalk for 3D collagen migration"

    Article Title: An extracellular matrix-specific GEF-GAP interaction regulates Rho GTPase crosstalk for 3D collagen migration

    Journal: Nature cell biology

    doi: 10.1038/ncb3026

    Fibrillar collagen activates βPix through α 2 β 1 integrin, leading to a critical dephosphorylation at T526 through PP2A. ( a ) Loss of focal adhesion localization is a read-out of differential βPix function on fibrillar collagen ( Fig. 1c ). Dishes were coated with monoclonal integrin antibodies targeting β 1 (9EG7), α 5 (mAb 16), or α 2 (P1E6) to mimic integrin ligation. GFP-βPix knockdown/rescue cells were plated on the dishes and assayed for focal adhesion localization (red; yellow in overlay). Ligation of α 2 results in a dramatic loss in GFP-βPix (grayscale) localization at paxillin (red)-containing adhesions with no changes in overall focal adhesion profile. Scale bars, 25 μm. ( b ) Western blot of KDR-WT GFP-βPix immunoprecipitated from knockdown/rescue cells migrating on fibronectin or fibrillar collagen for phospho-threonine showed a decrease in phosphorylation levels during migration on collagen. Immunoprecipitation of KDR-T526A βPix showed no change in phospho-threonine between FN and FIB COL, highlighting the functional importance of this residue. ( c ) We generated phospho-mimetic (T526E) and phospho-null (T526A) mutant βPix knockdown/rescue cells and assayed their morphology in 3D collagen. T526E βPix was insufficient to rescue the morphological and hypercontractile phenotype of βPix knockdown (collagen fibers, red, reflection microscopy). T526A mutants efficiently rescued the βPix morphological and contractile defects. Scale bars, 25 μm. ( d ) Quantification of cell velocity in βPix knockdown/rescue phosphovariants in 3D collagen. n = 25, 24, 22, and 22 cells for βPix sh#2, WT, T526E, and T526A were assessed across three independent experiments (mean ± s.e.m., one-way ANOVA with Bonferroni multiple comparisons correction). ( e ) GFP-βPix was immunoprecipitated from HFFs expressing knockdown/rescue phosphovariants at Thr526 migrating on fibrillar collagen. We find that phosphorylationmimetic (T526E) inhibits binding to srGAP1, but not Cdc42. ( f ) Immunoprecipitation of GFP-βPix from βPix knockdown/rescue cells migrating on fibronectin versus fibrillar collagen identified a collagen-specific interaction between βPix and PP2A regulatory subunit A α isoform (PPP2R1A). ( g ) GFP-βPix knockdown/rescue fibroblasts migrating on fibrillar collagen were treated with NS or PPP2R1A siRNA #1. We observed that knockdown or inhibition ( Supplementary Fig. 5g ) of PPP2R1A increased phosphothreonine levels on βPix during migration on collagen. ( h ) Summary model of the collagen-specific role of βPix during migration in fibrillar collagen environments. All western blots are representative of at least three independent experiments. Statistical source data can be found in Supplementary Table 2 , *** P
    Figure Legend Snippet: Fibrillar collagen activates βPix through α 2 β 1 integrin, leading to a critical dephosphorylation at T526 through PP2A. ( a ) Loss of focal adhesion localization is a read-out of differential βPix function on fibrillar collagen ( Fig. 1c ). Dishes were coated with monoclonal integrin antibodies targeting β 1 (9EG7), α 5 (mAb 16), or α 2 (P1E6) to mimic integrin ligation. GFP-βPix knockdown/rescue cells were plated on the dishes and assayed for focal adhesion localization (red; yellow in overlay). Ligation of α 2 results in a dramatic loss in GFP-βPix (grayscale) localization at paxillin (red)-containing adhesions with no changes in overall focal adhesion profile. Scale bars, 25 μm. ( b ) Western blot of KDR-WT GFP-βPix immunoprecipitated from knockdown/rescue cells migrating on fibronectin or fibrillar collagen for phospho-threonine showed a decrease in phosphorylation levels during migration on collagen. Immunoprecipitation of KDR-T526A βPix showed no change in phospho-threonine between FN and FIB COL, highlighting the functional importance of this residue. ( c ) We generated phospho-mimetic (T526E) and phospho-null (T526A) mutant βPix knockdown/rescue cells and assayed their morphology in 3D collagen. T526E βPix was insufficient to rescue the morphological and hypercontractile phenotype of βPix knockdown (collagen fibers, red, reflection microscopy). T526A mutants efficiently rescued the βPix morphological and contractile defects. Scale bars, 25 μm. ( d ) Quantification of cell velocity in βPix knockdown/rescue phosphovariants in 3D collagen. n = 25, 24, 22, and 22 cells for βPix sh#2, WT, T526E, and T526A were assessed across three independent experiments (mean ± s.e.m., one-way ANOVA with Bonferroni multiple comparisons correction). ( e ) GFP-βPix was immunoprecipitated from HFFs expressing knockdown/rescue phosphovariants at Thr526 migrating on fibrillar collagen. We find that phosphorylationmimetic (T526E) inhibits binding to srGAP1, but not Cdc42. ( f ) Immunoprecipitation of GFP-βPix from βPix knockdown/rescue cells migrating on fibronectin versus fibrillar collagen identified a collagen-specific interaction between βPix and PP2A regulatory subunit A α isoform (PPP2R1A). ( g ) GFP-βPix knockdown/rescue fibroblasts migrating on fibrillar collagen were treated with NS or PPP2R1A siRNA #1. We observed that knockdown or inhibition ( Supplementary Fig. 5g ) of PPP2R1A increased phosphothreonine levels on βPix during migration on collagen. ( h ) Summary model of the collagen-specific role of βPix during migration in fibrillar collagen environments. All western blots are representative of at least three independent experiments. Statistical source data can be found in Supplementary Table 2 , *** P

    Techniques Used: De-Phosphorylation Assay, Ligation, Western Blot, Immunoprecipitation, Migration, Functional Assay, Generated, Mutagenesis, Microscopy, Expressing, Binding Assay, Inhibition

    A collagen-specific GEF/GAP interaction between βPix and srGAP1 regulates suppression of RhoA activity. ( a ) Immunoprecipitation of GFP-βPix from βPix knockdown/rescue HFFs migrating on fibronectin (FN) versus fibrillar collagen (FIB COL) identifies a collagen-specific GEF/GAP interaction between βPix and srGAP1. ( b ) Concurrent decreased association of βPix with known effector Pak1 when migrating on fibrillar collagen. Blots are representative of three independent experiments. ( c ) RhoA activity determined by GST-RBD binding from NS and srGAP1 siRNA-treated HFFs migrating on fibronectin or fibrillar collagen environments. ( d ) Quantification of bands again revealed a 40-60% collagen-specific increase in RhoA activity after loss of srGAP1 (mean ± s.e.m., n = 3 independent western blots, t -tests). ( e ) srGAP1 knockdown HFFs were cultured overnight in 3D collagen gels. Fixation and labeling with Alexa488-phaloidin revealed a rounded, protrusive (white arrowheads) morphology akin to βPix knockdown. Similarly, srGAP1 knockdown fibroblasts severely alter collagen fiber arrangement (red, reflection microscopy) adjacent to the cell. Hole in matrix marked by white asterisk; scale bar, 25 μm. ( f ) Quantification of cell protrusions in cells treated with srGAP1 siRNA in 3D collagen. n = 36, 36, and 24 cells for NS, βPix si#1, and srGAP1 si#1 were assessed across three independent experiments (mean ± s.e.m., one-way ANOVA with Bonferroni multiple comparisons correction). ( g ) Quantification of cell velocity in cells treated with srGAP1 siRNA in 3D collagen. n = 25, 24, and 21 cells for NS, βPix si#1, and srGAP1 si#1 were assessed across three independent experiments (mean ± s.e.m., one-way ANOVA with Bonferroni multiple comparisons correction). Statistical source data can be found in Supplementary Table 2 , *** P
    Figure Legend Snippet: A collagen-specific GEF/GAP interaction between βPix and srGAP1 regulates suppression of RhoA activity. ( a ) Immunoprecipitation of GFP-βPix from βPix knockdown/rescue HFFs migrating on fibronectin (FN) versus fibrillar collagen (FIB COL) identifies a collagen-specific GEF/GAP interaction between βPix and srGAP1. ( b ) Concurrent decreased association of βPix with known effector Pak1 when migrating on fibrillar collagen. Blots are representative of three independent experiments. ( c ) RhoA activity determined by GST-RBD binding from NS and srGAP1 siRNA-treated HFFs migrating on fibronectin or fibrillar collagen environments. ( d ) Quantification of bands again revealed a 40-60% collagen-specific increase in RhoA activity after loss of srGAP1 (mean ± s.e.m., n = 3 independent western blots, t -tests). ( e ) srGAP1 knockdown HFFs were cultured overnight in 3D collagen gels. Fixation and labeling with Alexa488-phaloidin revealed a rounded, protrusive (white arrowheads) morphology akin to βPix knockdown. Similarly, srGAP1 knockdown fibroblasts severely alter collagen fiber arrangement (red, reflection microscopy) adjacent to the cell. Hole in matrix marked by white asterisk; scale bar, 25 μm. ( f ) Quantification of cell protrusions in cells treated with srGAP1 siRNA in 3D collagen. n = 36, 36, and 24 cells for NS, βPix si#1, and srGAP1 si#1 were assessed across three independent experiments (mean ± s.e.m., one-way ANOVA with Bonferroni multiple comparisons correction). ( g ) Quantification of cell velocity in cells treated with srGAP1 siRNA in 3D collagen. n = 25, 24, and 21 cells for NS, βPix si#1, and srGAP1 si#1 were assessed across three independent experiments (mean ± s.e.m., one-way ANOVA with Bonferroni multiple comparisons correction). Statistical source data can be found in Supplementary Table 2 , *** P

    Techniques Used: Activity Assay, Immunoprecipitation, Binding Assay, Western Blot, Cell Culture, Labeling, Microscopy

    28) Product Images from "miR-29a promotes hepatitis B virus replication and expression by targeting SMARCE1 in hepatoma carcinoma"

    Article Title: miR-29a promotes hepatitis B virus replication and expression by targeting SMARCE1 in hepatoma carcinoma

    Journal: World Journal of Gastroenterology

    doi: 10.3748/wjg.v23.i25.4569

    SMARCE1 is a target of miR-29a ( n = 3). A: Wild-type (WT) and mutant (MUT) 3’-UTR binding sites are shown. The mutated bases are labelled with a horizontal line; B: Relative luciferase activity was measured in HEK293 cells co-transfected WT or MUT SMARCE1 3’-UTR with miR-17 mimic or miR-control; C-E: The relative mRNA (C) and protein (D and E) levels of SMARCE1 were measured in HepG2.2.15 cells transfected with miR-29a mimics or anti-miR-29a. Data represent the mean ± SD. a P
    Figure Legend Snippet: SMARCE1 is a target of miR-29a ( n = 3). A: Wild-type (WT) and mutant (MUT) 3’-UTR binding sites are shown. The mutated bases are labelled with a horizontal line; B: Relative luciferase activity was measured in HEK293 cells co-transfected WT or MUT SMARCE1 3’-UTR with miR-17 mimic or miR-control; C-E: The relative mRNA (C) and protein (D and E) levels of SMARCE1 were measured in HepG2.2.15 cells transfected with miR-29a mimics or anti-miR-29a. Data represent the mean ± SD. a P

    Techniques Used: Mutagenesis, Binding Assay, Luciferase, Activity Assay, Transfection

    Effect of SMARCE1 on hepatitis B virus replication and expression ( n = 3). A: HepG2.2.15 cells were transfected with pcDNA-control, pcDNA-SMARCE1, si-control, or si-SMARCE1 for 24 h; B and E: Western blotting was performed to detect the level of SMARCE1 after pcDNA-SMARCE1 or si-SMARCE1 transfection; C and D: qPCR and Southern blotting analysis showed that SMARCE1 overexpression significantly reduced the HBV DNA replication and SMARCE1 knockdown significantly increased the HBV DNA replication; F and G: ELISA assay showed that SMARCE1 overexpression significantly decreased the levels of HBsAg and HBeAg; H and I: Endogenous SMARCE1 inhibition by si-SMARCE1 significantly promoted the levels of HBsAg and HBeAg. a P
    Figure Legend Snippet: Effect of SMARCE1 on hepatitis B virus replication and expression ( n = 3). A: HepG2.2.15 cells were transfected with pcDNA-control, pcDNA-SMARCE1, si-control, or si-SMARCE1 for 24 h; B and E: Western blotting was performed to detect the level of SMARCE1 after pcDNA-SMARCE1 or si-SMARCE1 transfection; C and D: qPCR and Southern blotting analysis showed that SMARCE1 overexpression significantly reduced the HBV DNA replication and SMARCE1 knockdown significantly increased the HBV DNA replication; F and G: ELISA assay showed that SMARCE1 overexpression significantly decreased the levels of HBsAg and HBeAg; H and I: Endogenous SMARCE1 inhibition by si-SMARCE1 significantly promoted the levels of HBsAg and HBeAg. a P

    Techniques Used: Expressing, Transfection, Western Blot, Real-time Polymerase Chain Reaction, Southern Blot, Over Expression, Enzyme-linked Immunosorbent Assay, Inhibition

    miR-29a promotes hepatitis B virus replication and expression by targeted regulation of SMARCE1 ( n = 3). HepG2.2.15 cells were transfected with miR-control or miR-29a mimics or co-transfected miR-29a mimics with pcDNA-control or pcDNA-SMARCE1. A and B: Restored expression of SMARCE1 by pcDNA-SMARCE1 relieved the increased HBV replication by miR-29a overexpression; C and D: Restored expression of SMARCE1 by pcDNA-SMARCE1 reversed the increased HBsAg and HBeAg expression by miR-29a overexpression. Data represent the mean ± SD. a P
    Figure Legend Snippet: miR-29a promotes hepatitis B virus replication and expression by targeted regulation of SMARCE1 ( n = 3). HepG2.2.15 cells were transfected with miR-control or miR-29a mimics or co-transfected miR-29a mimics with pcDNA-control or pcDNA-SMARCE1. A and B: Restored expression of SMARCE1 by pcDNA-SMARCE1 relieved the increased HBV replication by miR-29a overexpression; C and D: Restored expression of SMARCE1 by pcDNA-SMARCE1 reversed the increased HBsAg and HBeAg expression by miR-29a overexpression. Data represent the mean ± SD. a P

    Techniques Used: Expressing, Transfection, Over Expression

    Expression of miR-29a and SMARCE1 in hepatitis B virus-infected HepG2.2.15 cells ( n = 3). A and B: qRT-PCR analysis showed the expression levels of miR-29a and SMARCE1 in HepG2.2.15 cells and HepG2 cells (NC); C and D: The protein level of SMARCE1 was detected and quantified by western blotting of HepG2.2.15 cells and HepG2 cells (NC). Data represent the mean ± SD. a P
    Figure Legend Snippet: Expression of miR-29a and SMARCE1 in hepatitis B virus-infected HepG2.2.15 cells ( n = 3). A and B: qRT-PCR analysis showed the expression levels of miR-29a and SMARCE1 in HepG2.2.15 cells and HepG2 cells (NC); C and D: The protein level of SMARCE1 was detected and quantified by western blotting of HepG2.2.15 cells and HepG2 cells (NC). Data represent the mean ± SD. a P

    Techniques Used: Expressing, Infection, Quantitative RT-PCR, Western Blot

    29) Product Images from "Opposing Growth Regulatory Roles of Protein Kinase D Isoforms in Human Keratinocytes *"

    Article Title: Opposing Growth Regulatory Roles of Protein Kinase D Isoforms in Human Keratinocytes *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M115.643742

    Divergence of PKD signaling between mouse and human KCs. A , protein lysates of subconfluent ( P ) and postconfluent ( D ) cultures of NHKC were analyzed by immunoblotting using antibodies specific to PKD3, PKD2, and markers of KC differentiation including
    Figure Legend Snippet: Divergence of PKD signaling between mouse and human KCs. A , protein lysates of subconfluent ( P ) and postconfluent ( D ) cultures of NHKC were analyzed by immunoblotting using antibodies specific to PKD3, PKD2, and markers of KC differentiation including

    Techniques Used:

    PKD3 is required for normal proliferation and differentiation in regenerating epidermis. A–D , organotypic epidermal tissue regenerated from NHKCs expressing shRNA against PKD2, PKD3, and a Scr control. Tissue sections were analyzed by histology
    Figure Legend Snippet: PKD3 is required for normal proliferation and differentiation in regenerating epidermis. A–D , organotypic epidermal tissue regenerated from NHKCs expressing shRNA against PKD2, PKD3, and a Scr control. Tissue sections were analyzed by histology

    Techniques Used: Expressing, shRNA

    Opposing growth regulatory functions of PKD2 and PKD3 in human KCs. A , NHKCs were transduced with lentiviruses encoding shRNA targeted to PKD2, PKD3, or a scramble ( Scr ) shRNA. Cell lysates were prepared from subconfluent ( P ) and postconfluent ( D ) cultures
    Figure Legend Snippet: Opposing growth regulatory functions of PKD2 and PKD3 in human KCs. A , NHKCs were transduced with lentiviruses encoding shRNA targeted to PKD2, PKD3, or a scramble ( Scr ) shRNA. Cell lysates were prepared from subconfluent ( P ) and postconfluent ( D ) cultures

    Techniques Used: Transduction, shRNA

    30) Product Images from "USP9X counteracts differential ubiquitination of NPHP5 by MARCH7 and BBS11 to regulate ciliogenesis"

    Article Title: USP9X counteracts differential ubiquitination of NPHP5 by MARCH7 and BBS11 to regulate ciliogenesis

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1006791

    NPHP5 is K48 ubiquitinated by MARCH7. A) Flag or Flag-NPHP5 was co-expressed with Myc or Myc-MARCH7 in HEK293 cells. Lysates were immunoprecipitated with anti-Myc antibody and Western blotted with the indicated antibodies. IN, input. B) Myc, Myc-MARCH7 wild type or catalytically inactive mutant (WI) was co-expressed with Flag-NPHP5 and HA-Ub in HEK293 cells. Lysates were immunoprecipitated with anti-Flag antibody in 1% SDS and Western blotted with the indicated antibodies. IN, input. C) HEK293 cells were transfected with Myc, Myc-MARCH7 wild type or mutant. Lysates were Western blotted with the indicated antibodies. β actin was used as a loading control. D) HEK293 cells were transfected with control (NS) or MARCH7 siRNA. Lysates were Western blotted with the indicated antibodies. β actin was used as a loading control. E) Cycling RPE-1 cells expressing an irrelevant Myc tagged protein (control) or Myc-MARCH7 were stained with Myc (violet), NPHP5 (red) and γ-tubulin (green). F-G) An irrelevant Myc tagged protein (control) or Myc-MARCH7 was expressed in quiescent F) or cycling G) RPE-1 cells. ( F , top; G , left) Cells were stained with Myc and glutamylated tubulin (GT335) and the percentage of Myc positive cells with cilia was determined. ( F , bottom; G right) Lysates were Western blotted with the indicated antibodies. β actin was used as a loading control. Numbers indicate total MARCH7 levels relative to control. H) The percentage of RPE-1 cells with centrosomal MARCH7 staining across the cell cycle is presented. I) The percentage of cycling RPE-1 cells with centrosomal MARCH7 staining after transfection with control (NS) or USP9X siRNA is presented. J) HEK293 cells were transfected with control (NS) or USP9X siRNA and plasmids expressing Myc-MARCH7 and Flag-NPHP5. Lysates were immunoprecipitated with anti-Myc antibody and Western blotted with the indicated antibodies. IN, input. In F-I) at least 100 cells were counted per condition, and error bars represent average of three independent experiments. Asterisks indicate non-specific bands.
    Figure Legend Snippet: NPHP5 is K48 ubiquitinated by MARCH7. A) Flag or Flag-NPHP5 was co-expressed with Myc or Myc-MARCH7 in HEK293 cells. Lysates were immunoprecipitated with anti-Myc antibody and Western blotted with the indicated antibodies. IN, input. B) Myc, Myc-MARCH7 wild type or catalytically inactive mutant (WI) was co-expressed with Flag-NPHP5 and HA-Ub in HEK293 cells. Lysates were immunoprecipitated with anti-Flag antibody in 1% SDS and Western blotted with the indicated antibodies. IN, input. C) HEK293 cells were transfected with Myc, Myc-MARCH7 wild type or mutant. Lysates were Western blotted with the indicated antibodies. β actin was used as a loading control. D) HEK293 cells were transfected with control (NS) or MARCH7 siRNA. Lysates were Western blotted with the indicated antibodies. β actin was used as a loading control. E) Cycling RPE-1 cells expressing an irrelevant Myc tagged protein (control) or Myc-MARCH7 were stained with Myc (violet), NPHP5 (red) and γ-tubulin (green). F-G) An irrelevant Myc tagged protein (control) or Myc-MARCH7 was expressed in quiescent F) or cycling G) RPE-1 cells. ( F , top; G , left) Cells were stained with Myc and glutamylated tubulin (GT335) and the percentage of Myc positive cells with cilia was determined. ( F , bottom; G right) Lysates were Western blotted with the indicated antibodies. β actin was used as a loading control. Numbers indicate total MARCH7 levels relative to control. H) The percentage of RPE-1 cells with centrosomal MARCH7 staining across the cell cycle is presented. I) The percentage of cycling RPE-1 cells with centrosomal MARCH7 staining after transfection with control (NS) or USP9X siRNA is presented. J) HEK293 cells were transfected with control (NS) or USP9X siRNA and plasmids expressing Myc-MARCH7 and Flag-NPHP5. Lysates were immunoprecipitated with anti-Myc antibody and Western blotted with the indicated antibodies. IN, input. In F-I) at least 100 cells were counted per condition, and error bars represent average of three independent experiments. Asterisks indicate non-specific bands.

    Techniques Used: Immunoprecipitation, Western Blot, Mutagenesis, Transfection, Expressing, Staining

    NPHP5 is deubiquitinated by USP9X. A) In vitro deubiquitination assays were performed by adding poly (HA)-ubiquitinated NPHP5 as a substrate to GFP, GFP-USP9X wild type or mutant (C1566S) bound to beads. Poly (HA)-ubiquitinated NPHP5 and GFP proteins in the reaction mixture were detected by immunoblotting with anti-HA and anti-GFP antibodies, respectively. IN, input. B, D) HEK293 cells were transfected with control (NS) or USP9X siRNA and plasmids expressing Flag-NPHP5 and HA-Ub. Lysates were subjected to immunoprecipitation with anti-Flag antibody in 1% SDS and Western blotted with the indicated antibodies. IN, input. C) HEK293 cells were transfected with GFP, GFP-USP9X wild type or mutant (C1566S), Flag-NPHP5 and HA-Ub. Lysates were immunoprecipitated with anti-Flag antibody in 1% SDS and Western blotted with the indicated antibodies. IN, input. Asterisks indicate non-specific bands.
    Figure Legend Snippet: NPHP5 is deubiquitinated by USP9X. A) In vitro deubiquitination assays were performed by adding poly (HA)-ubiquitinated NPHP5 as a substrate to GFP, GFP-USP9X wild type or mutant (C1566S) bound to beads. Poly (HA)-ubiquitinated NPHP5 and GFP proteins in the reaction mixture were detected by immunoblotting with anti-HA and anti-GFP antibodies, respectively. IN, input. B, D) HEK293 cells were transfected with control (NS) or USP9X siRNA and plasmids expressing Flag-NPHP5 and HA-Ub. Lysates were subjected to immunoprecipitation with anti-Flag antibody in 1% SDS and Western blotted with the indicated antibodies. IN, input. C) HEK293 cells were transfected with GFP, GFP-USP9X wild type or mutant (C1566S), Flag-NPHP5 and HA-Ub. Lysates were immunoprecipitated with anti-Flag antibody in 1% SDS and Western blotted with the indicated antibodies. IN, input. Asterisks indicate non-specific bands.

    Techniques Used: In Vitro, Mutagenesis, Transfection, Expressing, Immunoprecipitation, Western Blot

    NPHP5 is a low turnover protein which becomes K63 ubiquitinated in mitosis. A-B) U2OS A) and RPE-1 B) lysates from different cell cycle phases were immunoprecipitated with anti-NPHP5 (IRCM1) antibody and Western blotted with the indicated antibodies. As, asynchronous; IN, input. C) RPE-1 cells in different stages of the cell cycle were processed for immunofluorescence with anti-USP9X (red) and anti-centrin (green) antibodies. DNA was stained with DAPI (blue). D-E) U2OS cells expressing Flag-NPHP5 and HA-Ub were synchronized in different cell cycle phases or left asynchronized (As). Lysates were immunoprecipitated with anti-Flag antibody in 1% SDS and Western blotted with the indicated antibodies. IN, input. Asterisks indicate non-specific bands.
    Figure Legend Snippet: NPHP5 is a low turnover protein which becomes K63 ubiquitinated in mitosis. A-B) U2OS A) and RPE-1 B) lysates from different cell cycle phases were immunoprecipitated with anti-NPHP5 (IRCM1) antibody and Western blotted with the indicated antibodies. As, asynchronous; IN, input. C) RPE-1 cells in different stages of the cell cycle were processed for immunofluorescence with anti-USP9X (red) and anti-centrin (green) antibodies. DNA was stained with DAPI (blue). D-E) U2OS cells expressing Flag-NPHP5 and HA-Ub were synchronized in different cell cycle phases or left asynchronized (As). Lysates were immunoprecipitated with anti-Flag antibody in 1% SDS and Western blotted with the indicated antibodies. IN, input. Asterisks indicate non-specific bands.

    Techniques Used: Immunoprecipitation, Western Blot, Immunofluorescence, Staining, Expressing

    Effects of deregulating USP9X on NPHP5. A) RPE-1 cells transfected with control (NS) or USP9X siRNA and stained with antibodies against USP9X or NPHP5 (red) and centrin (green). DNA was stained with DAPI (blue). B) HEK293 cells were transfected with control (NS) or USP9X siRNA. Lysates were Western blotted with the indicated antibodies. β actin was used as a loading control. Numbers indicate NPHP5 protein levels relative to control. C) HEK293 cells were transfected with control (NS) or USP9X siRNA and treated with or without MG132. Lysates were Western blotted with the indicated antibodies. β actin was used as a loading control. Numbers indicate NPHP5 protein levels relative to control without MG132 treatment. D) HEK293 cells were transfected with control (NS) or USP9X siRNA and treated with cycloheximide for the indicated number of hours (hrs). Lysates were Western blotted with the indicated antibodies. β actin was used as a loading control. Numbers indicate NPHP5 protein levels relative to control at time zero. E) HEK293 cells were transfected with GFP, GFP-USP9X wild type or mutant (C1566S). Lysates were Western blotted with the indicated antibodies. β actin was used as a loading control. F) RPE-1 cells were transfected with control (NS) or USP9X siRNA, induced to quiescence, and stained with antibodies against USP9X (red) and glutamylated tubulin (GT335; green), and with DAPI (blue). G-H) The percentage of control (NS) or USP9X siRNA-treated cycling or quiescent RPE-1 cells with cilia was determined. Cilia were stained with glutamylated tubulin (GT335), acetylated tubulin (Ac-Tub) or IFT88. I) Fluorescence-activated cell sorting analysis of cycling RPE-1 cells treated with control (NS) or USP9X siRNA. J-K ) The percentage of control (NS) or USP9X siRNA-treated RPE-1 cells stained positive for Ki67 was determined. L) RPE-1 cells transfected with control (NS) or USP9X siRNA and plasmid expressing GFP or GFP-NPHP5 were induced to quiescence. The percentage of GFP positive cells with cilia was determined. In G, H, J, K, L) , at least 100 cells were counted per condition, and error bars represent average of three independent experiments.
    Figure Legend Snippet: Effects of deregulating USP9X on NPHP5. A) RPE-1 cells transfected with control (NS) or USP9X siRNA and stained with antibodies against USP9X or NPHP5 (red) and centrin (green). DNA was stained with DAPI (blue). B) HEK293 cells were transfected with control (NS) or USP9X siRNA. Lysates were Western blotted with the indicated antibodies. β actin was used as a loading control. Numbers indicate NPHP5 protein levels relative to control. C) HEK293 cells were transfected with control (NS) or USP9X siRNA and treated with or without MG132. Lysates were Western blotted with the indicated antibodies. β actin was used as a loading control. Numbers indicate NPHP5 protein levels relative to control without MG132 treatment. D) HEK293 cells were transfected with control (NS) or USP9X siRNA and treated with cycloheximide for the indicated number of hours (hrs). Lysates were Western blotted with the indicated antibodies. β actin was used as a loading control. Numbers indicate NPHP5 protein levels relative to control at time zero. E) HEK293 cells were transfected with GFP, GFP-USP9X wild type or mutant (C1566S). Lysates were Western blotted with the indicated antibodies. β actin was used as a loading control. F) RPE-1 cells were transfected with control (NS) or USP9X siRNA, induced to quiescence, and stained with antibodies against USP9X (red) and glutamylated tubulin (GT335; green), and with DAPI (blue). G-H) The percentage of control (NS) or USP9X siRNA-treated cycling or quiescent RPE-1 cells with cilia was determined. Cilia were stained with glutamylated tubulin (GT335), acetylated tubulin (Ac-Tub) or IFT88. I) Fluorescence-activated cell sorting analysis of cycling RPE-1 cells treated with control (NS) or USP9X siRNA. J-K ) The percentage of control (NS) or USP9X siRNA-treated RPE-1 cells stained positive for Ki67 was determined. L) RPE-1 cells transfected with control (NS) or USP9X siRNA and plasmid expressing GFP or GFP-NPHP5 were induced to quiescence. The percentage of GFP positive cells with cilia was determined. In G, H, J, K, L) , at least 100 cells were counted per condition, and error bars represent average of three independent experiments.

    Techniques Used: Transfection, Staining, Western Blot, Mutagenesis, Fluorescence, FACS, Plasmid Preparation, Expressing

    NPHP5 recruits USP9X to the centrosome. A) HEK293 cells were transfected with control (NS) and NPHP5 siRNA. Lysates were Western blotted with the indicated antibodies. β actin was used as a loading control. B) RPE-1 cells transfected with control (NS) and NPHP5 siRNA were stained with anti-NPHP5 or anti-USP9X (red) and anti-centrin (green) antibodies. DNA was stained with DAPI (blue).
    Figure Legend Snippet: NPHP5 recruits USP9X to the centrosome. A) HEK293 cells were transfected with control (NS) and NPHP5 siRNA. Lysates were Western blotted with the indicated antibodies. β actin was used as a loading control. B) RPE-1 cells transfected with control (NS) and NPHP5 siRNA were stained with anti-NPHP5 or anti-USP9X (red) and anti-centrin (green) antibodies. DNA was stained with DAPI (blue).

    Techniques Used: Transfection, Western Blot, Staining

    NPHP5 directly interacts with USP9X. A) Flag or Flag-NPHP5 was expressed in HEK293 cells. Lysates were immunoprecipitated with anti-Flag antibody and Western blotted with the indicated antibodies. IN, input. B) (Top) IRCM1 and IRCM2 are two anti-NPHP5 antibodies raised against the C- and the N-terminus of NPHP5, respectively. Grey box, calmodulin-binding domain; white box, coiled-coil domain; black box; Cep290-binding domain. (Bottom) HEK293 lysates were immunoprecipitated with anti-Flag (control), anti-NPHP5 (IRCM1 or IRCM2) or anti-USP9X antibody and Western blotted with the indicated antibodies. IN, input. C) The ability of full-length NPHP5 (1-598(FL)) and various NPHP5 truncates to interact with USP9X is presented. D) Flag (control) or the indicated fragment of Flag-tagged NPHP5 was expressed in HEK293 cells. Lysates were immunoprecipitated with anti-Flag antibody and Western blotted with the indicated antibodies. IN, input. Asterisks indicate bands corresponding to the expected recombinant proteins. E) Purified GFP, GFP-USP9X wild type or catalytically inactive mutant (C1566S) bound to beads was mixed with purified Flag-NPHP5. Proteins recovered on beads were analyzed by Western blotting with the indicated antibodies. IN, input.
    Figure Legend Snippet: NPHP5 directly interacts with USP9X. A) Flag or Flag-NPHP5 was expressed in HEK293 cells. Lysates were immunoprecipitated with anti-Flag antibody and Western blotted with the indicated antibodies. IN, input. B) (Top) IRCM1 and IRCM2 are two anti-NPHP5 antibodies raised against the C- and the N-terminus of NPHP5, respectively. Grey box, calmodulin-binding domain; white box, coiled-coil domain; black box; Cep290-binding domain. (Bottom) HEK293 lysates were immunoprecipitated with anti-Flag (control), anti-NPHP5 (IRCM1 or IRCM2) or anti-USP9X antibody and Western blotted with the indicated antibodies. IN, input. C) The ability of full-length NPHP5 (1-598(FL)) and various NPHP5 truncates to interact with USP9X is presented. D) Flag (control) or the indicated fragment of Flag-tagged NPHP5 was expressed in HEK293 cells. Lysates were immunoprecipitated with anti-Flag antibody and Western blotted with the indicated antibodies. IN, input. Asterisks indicate bands corresponding to the expected recombinant proteins. E) Purified GFP, GFP-USP9X wild type or catalytically inactive mutant (C1566S) bound to beads was mixed with purified Flag-NPHP5. Proteins recovered on beads were analyzed by Western blotting with the indicated antibodies. IN, input.

    Techniques Used: Immunoprecipitation, Western Blot, Binding Assay, Recombinant, Purification, Mutagenesis

    Model depicting the role of ubiquitination and deubiquitination in controlling NPHP5-mediated ciliogenesis. (Top left) In the G0/G1/S phase, a pool of cytoplasmic USP9X is recruited to the centrosome by NPHP5. MARCH7 is held in the cytoplasm by USP9X, whereas BBS11 is localized to the centrosome. Because centrosomal USP9X actively deubiquitinates NPHP5, NPHP5 is stabilized and cilia assembly is favoured. (Top right) In the G2/M phase, USP9X dissociation from the centrosome makes NPHP5 prone to ubiquitination. NPHP5 undergoes K63-ubiquitination by BBS11 and becomes delocalized, and cilia disassembly is favoured. MARCH7 is sequestered by USP9X in the cytoplasm at G2. MARCH7 goes to the centrosome in mitosis, but NPHP5 is already delocalized. (Bottom) When USP9X is depleted or inhibited, NPHP5 is prone to ubiquitination. MARCH7 becomes aberrantly translocated to the centrosome wherein it K48-ubiquitinates NPHP5. BBS11 can K63-ubiquitinate NPHP5 at the same time. As a result, NPHP5 is degraded and delocalized, and cilia disassembly is favoured.
    Figure Legend Snippet: Model depicting the role of ubiquitination and deubiquitination in controlling NPHP5-mediated ciliogenesis. (Top left) In the G0/G1/S phase, a pool of cytoplasmic USP9X is recruited to the centrosome by NPHP5. MARCH7 is held in the cytoplasm by USP9X, whereas BBS11 is localized to the centrosome. Because centrosomal USP9X actively deubiquitinates NPHP5, NPHP5 is stabilized and cilia assembly is favoured. (Top right) In the G2/M phase, USP9X dissociation from the centrosome makes NPHP5 prone to ubiquitination. NPHP5 undergoes K63-ubiquitination by BBS11 and becomes delocalized, and cilia disassembly is favoured. MARCH7 is sequestered by USP9X in the cytoplasm at G2. MARCH7 goes to the centrosome in mitosis, but NPHP5 is already delocalized. (Bottom) When USP9X is depleted or inhibited, NPHP5 is prone to ubiquitination. MARCH7 becomes aberrantly translocated to the centrosome wherein it K48-ubiquitinates NPHP5. BBS11 can K63-ubiquitinate NPHP5 at the same time. As a result, NPHP5 is degraded and delocalized, and cilia disassembly is favoured.

    Techniques Used:

    31) Product Images from "Depletion of Nsd2-mediated histone H3K36 methylation impairs adipose tissue development and function"

    Article Title: Depletion of Nsd2-mediated histone H3K36 methylation impairs adipose tissue development and function

    Journal: Nature Communications

    doi: 10.1038/s41467-018-04127-6

    H3.3K36M targets H3K36 methyltransferase Nsd2 to inhibit adipogenesis and PPARγ target gene expression. a – e Immortalized brown preadipocytes were infected with lentiviral vector expressing control (Ctrl) or Nsd2 knockdown (KD) shRNAs, followed by adipogenesis assay until D7. Cells were collected at D0 and D2 for RNA-Seq. a Western blot of Nsd2 and histone methylations in preadipocytes. RbBP5 and histone H3 were used as loading controls. b Oil Red O staining at D7 of adipogenesis. Scale bars = 30 μm. c Schematic identification of Nsd2-dependent and Nsd2-independent up-regulated genes at D2 of adipogenesis. The threshold for up-regulation or down-regulation is twofold. d GO analysis of gene groups defined in c . e Venn diagram depicting Nsd2-dependent (507) and K36M-sensitive (380) up-regulated genes at D2. f – h Nsd2 methyltransferase activity is required for adipogenesis. Nsd2 knockout (KO) preadipocytes were generated using CRISPR. Control (Ctrl) and Nsd2 KO cells were infected with retroviral vector expressing either WT, Y1092A/Y1179A mutant (DM1), or H1042G/Y1179A mutant (DM2) human NSD2, followed by adipogenesis assay. f Western blot of Nsd2 and histone methylations in preadipocytes. g Cells were stained with Oil Red O at D7 of adipogenesis. Scale bars = 30 μm. h qRT-PCR of Pparg and Cebpa expression at D0 and D7 of adipogenesis. i Nsd2 is required for ligand-induced PPARγ target gene expression. Ctrl or Nsd2 KD preadipocytes were infected with retroviral vector expressing PPARγ. Sub-confluent cells were treated with DMSO or 0.5 μM Rosi for 24 h, followed by qRT-PCR of Pparg and its target genes Cebpa , Cd36 , and Lpl . All qRT-PCR data are presented as means ± SEM. Three technical replicates from a single experiment were used
    Figure Legend Snippet: H3.3K36M targets H3K36 methyltransferase Nsd2 to inhibit adipogenesis and PPARγ target gene expression. a – e Immortalized brown preadipocytes were infected with lentiviral vector expressing control (Ctrl) or Nsd2 knockdown (KD) shRNAs, followed by adipogenesis assay until D7. Cells were collected at D0 and D2 for RNA-Seq. a Western blot of Nsd2 and histone methylations in preadipocytes. RbBP5 and histone H3 were used as loading controls. b Oil Red O staining at D7 of adipogenesis. Scale bars = 30 μm. c Schematic identification of Nsd2-dependent and Nsd2-independent up-regulated genes at D2 of adipogenesis. The threshold for up-regulation or down-regulation is twofold. d GO analysis of gene groups defined in c . e Venn diagram depicting Nsd2-dependent (507) and K36M-sensitive (380) up-regulated genes at D2. f – h Nsd2 methyltransferase activity is required for adipogenesis. Nsd2 knockout (KO) preadipocytes were generated using CRISPR. Control (Ctrl) and Nsd2 KO cells were infected with retroviral vector expressing either WT, Y1092A/Y1179A mutant (DM1), or H1042G/Y1179A mutant (DM2) human NSD2, followed by adipogenesis assay. f Western blot of Nsd2 and histone methylations in preadipocytes. g Cells were stained with Oil Red O at D7 of adipogenesis. Scale bars = 30 μm. h qRT-PCR of Pparg and Cebpa expression at D0 and D7 of adipogenesis. i Nsd2 is required for ligand-induced PPARγ target gene expression. Ctrl or Nsd2 KD preadipocytes were infected with retroviral vector expressing PPARγ. Sub-confluent cells were treated with DMSO or 0.5 μM Rosi for 24 h, followed by qRT-PCR of Pparg and its target genes Cebpa , Cd36 , and Lpl . All qRT-PCR data are presented as means ± SEM. Three technical replicates from a single experiment were used

    Techniques Used: Expressing, Infection, Plasmid Preparation, RNA Sequencing Assay, Western Blot, Staining, Activity Assay, Knock-Out, Generated, CRISPR, Mutagenesis, Quantitative RT-PCR

    32) Product Images from "Nuclear trafficking of the HIV-1 pre-integration complex depends on the ADAM10 intracellular domain"

    Article Title: Nuclear trafficking of the HIV-1 pre-integration complex depends on the ADAM10 intracellular domain

    Journal: Virology

    doi: 10.1016/j.virol.2014.02.006

    Colocalization and coimmunoprecipitation of the ADAM10 ICD and HIV-1 IN. U373 cells were co-cultured with the HIV-producing cell line, U1/HIV-1. Cells were stained with anti-IN and anti-ADAM10 ICD primary antibodies, followed by PLA secondary antibodies, and then fixed and imaged for immunofluorescence. Representative experiments are shown. (A) PLA results using an isotype-matched negative control anti-biotin antibody as the primary antibody. (B) Colocalization between the ADAM10 ICD and HIV-1 IN as revealed by PLA with punctate patterns of red fluorescence. Cell nuclei are stained with DAPI for contrast. (C) U373 cells were co-cultured for 6 h with an HIV-1 producing cell line, U1/HIV-1. Subsequently, cells were harvested and cytoplasmic fractions were collected. Co-immunoprecipitation was performed using an anti-IN mAb. Precipitates were separated by SDS-PAGE, transferred to a poly-vinyl membrane, and analyzed in Western blots. The membranes were cut into strips and probed with the indicated monoclonal antibodies. p75/LEDGF and HIV-1 IN were used as positive controls to confirm that whole PICs were precipitated. Actin and non-immune serum (NIS) were used as negative controls for non-specific precipitation of cellular proteins.
    Figure Legend Snippet: Colocalization and coimmunoprecipitation of the ADAM10 ICD and HIV-1 IN. U373 cells were co-cultured with the HIV-producing cell line, U1/HIV-1. Cells were stained with anti-IN and anti-ADAM10 ICD primary antibodies, followed by PLA secondary antibodies, and then fixed and imaged for immunofluorescence. Representative experiments are shown. (A) PLA results using an isotype-matched negative control anti-biotin antibody as the primary antibody. (B) Colocalization between the ADAM10 ICD and HIV-1 IN as revealed by PLA with punctate patterns of red fluorescence. Cell nuclei are stained with DAPI for contrast. (C) U373 cells were co-cultured for 6 h with an HIV-1 producing cell line, U1/HIV-1. Subsequently, cells were harvested and cytoplasmic fractions were collected. Co-immunoprecipitation was performed using an anti-IN mAb. Precipitates were separated by SDS-PAGE, transferred to a poly-vinyl membrane, and analyzed in Western blots. The membranes were cut into strips and probed with the indicated monoclonal antibodies. p75/LEDGF and HIV-1 IN were used as positive controls to confirm that whole PICs were precipitated. Actin and non-immune serum (NIS) were used as negative controls for non-specific precipitation of cellular proteins.

    Techniques Used: Cell Culture, Staining, Proximity Ligation Assay, Immunofluorescence, Negative Control, Fluorescence, Immunoprecipitation, SDS Page, Western Blot

    33) Product Images from "Specific MHC-I Peptides Are Induced Using PROTACs"

    Article Title: Specific MHC-I Peptides Are Induced Using PROTACs

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.02697

    PROTAC-induced BRD peptides across all time points and concentrations. (A) ). Map of specific PROTAC compounds that induced presentation of each peptide. (B) BRD protein family domain map, showing the relative locations of bromodomain 1 (BD1) and bromodomain 2 (BD2) as well as two conserved regions (A and B) within the protein sequence. JQ1 cooperatively binds and degrades BRD2 within BD1. JQ1-VHL cooperatively binds and degrades BRD3 and BRD4 within BD2. Observed peptides derived from BRD2, BRD3, and BRD2/3/4 are annotated over the BRD family protein structure, with colored boxes representing JQ1-CRBN (green), JQ1-VHL (blue), and JQ1-MDM2 (red) induced presentation.
    Figure Legend Snippet: PROTAC-induced BRD peptides across all time points and concentrations. (A) ). Map of specific PROTAC compounds that induced presentation of each peptide. (B) BRD protein family domain map, showing the relative locations of bromodomain 1 (BD1) and bromodomain 2 (BD2) as well as two conserved regions (A and B) within the protein sequence. JQ1 cooperatively binds and degrades BRD2 within BD1. JQ1-VHL cooperatively binds and degrades BRD3 and BRD4 within BD2. Observed peptides derived from BRD2, BRD3, and BRD2/3/4 are annotated over the BRD family protein structure, with colored boxes representing JQ1-CRBN (green), JQ1-VHL (blue), and JQ1-MDM2 (red) induced presentation.

    Techniques Used: Sequencing, Derivative Assay

    34) Product Images from "Activation of p53 signaling by MI-63 induces apoptosis in acute myelogenous leukemia cells"

    Article Title: Activation of p53 signaling by MI-63 induces apoptosis in acute myelogenous leukemia cells

    Journal: Leukemia & lymphoma

    doi: 10.3109/10428191003731325

    Effect of MI-63 on MDM4 m-RNA and protein levels.
    Figure Legend Snippet: Effect of MI-63 on MDM4 m-RNA and protein levels.

    Techniques Used:

    35) Product Images from "Translocation t(2;11) in CLL cells results in CXCR4/MAML2 fusion oncogene"

    Article Title: Translocation t(2;11) in CLL cells results in CXCR4/MAML2 fusion oncogene

    Journal: Blood

    doi: 10.1182/blood-2014-02-554675

    CXCR4/MAML2 fusion gene is expressed in CLL. (A) Top panel, Eco RI restriction map around the breakpoint sites on chromosomes 2 and 11. The green line represents the full-length Eco RI-digested fragment without any break; the light blue line represents
    Figure Legend Snippet: CXCR4/MAML2 fusion gene is expressed in CLL. (A) Top panel, Eco RI restriction map around the breakpoint sites on chromosomes 2 and 11. The green line represents the full-length Eco RI-digested fragment without any break; the light blue line represents

    Techniques Used:

    36) Product Images from "Patched-1 Proapoptotic Activity Is Downregulated by Modification of K1413 by the E3 Ubiquitin-Protein Ligase Itchy Homolog"

    Article Title: Patched-1 Proapoptotic Activity Is Downregulated by Modification of K1413 by the E3 Ubiquitin-Protein Ligase Itchy Homolog

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00960-14

    The proapoptotic C-terminal domain of PTCH1 interacts with AIP4 and WWP2. (A) Diagrammatic view and Western blot of the membrane-targeted GFP fusion constructs utilized for pulldown and mass spectrometry. AcGFP, acetylated GFP. (B) Whole-cell lysates
    Figure Legend Snippet: The proapoptotic C-terminal domain of PTCH1 interacts with AIP4 and WWP2. (A) Diagrammatic view and Western blot of the membrane-targeted GFP fusion constructs utilized for pulldown and mass spectrometry. AcGFP, acetylated GFP. (B) Whole-cell lysates

    Techniques Used: Western Blot, Construct, Mass Spectrometry

    Itch and WWP2 interact with PTCH1 through two PPXY motifs. HEK293T cells were transfected for 18 h with wild-type (WT) PTCH1-HA, PTCH1-HA mutated in the C-terminal domain PPXY motif (C-PPXA), PTCH1-HA mutated in the central loop PPXY motif (L-PPXA), or
    Figure Legend Snippet: Itch and WWP2 interact with PTCH1 through two PPXY motifs. HEK293T cells were transfected for 18 h with wild-type (WT) PTCH1-HA, PTCH1-HA mutated in the C-terminal domain PPXY motif (C-PPXA), PTCH1-HA mutated in the central loop PPXY motif (L-PPXA), or

    Techniques Used: Transfection

    37) Product Images from "MiT/ TFE factors control ER‐phagy via transcriptional regulation of FAM134B"

    Article Title: MiT/ TFE factors control ER‐phagy via transcriptional regulation of FAM134B

    Journal: The EMBO Journal

    doi: 10.15252/embj.2020105696

    FGF‐dependent regulation of IRS1‐PI3K signaling and of TFEB and TFE3 activity Western blot analysis of p‐P70S6K (T389), P70S6K, p‐4EBP1(S65), 4EBP1, p‐S6 ribosomal protein (S240/S242), S6 ribosomal protein, p‐AKT (S473), AKT proteins in RCS chondrocytes treated as indicated (FGF18: 50 ng/ml 16 h; Torin 1: 1 μM for 2 h; amino acid starvation (AA‐) for 50 min; amino acid re‐feeding (AA) : 3× for 20 min). Representative images of N = 3 biological replicates. β‐actin was used as a loading control. Western blot analysis of IRS1 and phospho‐IRS1 (S307) in chondrocytes treated with vehicle (5%ABS), FGF18 (50 ng/ml), and JNK inhibitor (50 μM) for indicated time. Filamin A was used as a loading control. Blot is representative of N = 3 independent experiments. KEGG pathway analysis of Quantsec gene expression analysis in RCS chondrocytes treated with vehicle (5% ABS) and FGF18 (50 ng/ml) overnight, showing upregulated (red) and downregulated (green) biological processes and cellular components. Schematic diagram showing qPCR primers used to analyze Fam134b isoform expressions. Arrows indicate the positions qPCR primer pairs used to detect Fam134b‐1 (black arrows), Fam134b‐2 (brown arrows), or both (turquoise arrows) Fam134b isoforms. Primer sequences are listed in Materials and Methods . TFEB (green) subcellular localization analysis in wild type (control) and FGFR3;4 KO chondrocytes treated with FGF18 (50 ng/ml) for 16 h. Torin 1 was used at 1 μM for 2 h as positive control of TFEB nuclear translocation. Nuclei were stained with DAPI (blue). Scale bar 15 μm. Quantification is shown in Fig 3 A. Chromatin immunoprecipitation experiment in TFEB‐WT overexpressing cells treated with FGF18 (50 ng/ml) for 16 h, showing enrichment of TFEB binding on Mucolipin‐1 promoter upon FGF18 treatment. N = 3 biological replicates. Mean ± standard error (sd). Student's unpaired t ‐test ** P
    Figure Legend Snippet: FGF‐dependent regulation of IRS1‐PI3K signaling and of TFEB and TFE3 activity Western blot analysis of p‐P70S6K (T389), P70S6K, p‐4EBP1(S65), 4EBP1, p‐S6 ribosomal protein (S240/S242), S6 ribosomal protein, p‐AKT (S473), AKT proteins in RCS chondrocytes treated as indicated (FGF18: 50 ng/ml 16 h; Torin 1: 1 μM for 2 h; amino acid starvation (AA‐) for 50 min; amino acid re‐feeding (AA) : 3× for 20 min). Representative images of N = 3 biological replicates. β‐actin was used as a loading control. Western blot analysis of IRS1 and phospho‐IRS1 (S307) in chondrocytes treated with vehicle (5%ABS), FGF18 (50 ng/ml), and JNK inhibitor (50 μM) for indicated time. Filamin A was used as a loading control. Blot is representative of N = 3 independent experiments. KEGG pathway analysis of Quantsec gene expression analysis in RCS chondrocytes treated with vehicle (5% ABS) and FGF18 (50 ng/ml) overnight, showing upregulated (red) and downregulated (green) biological processes and cellular components. Schematic diagram showing qPCR primers used to analyze Fam134b isoform expressions. Arrows indicate the positions qPCR primer pairs used to detect Fam134b‐1 (black arrows), Fam134b‐2 (brown arrows), or both (turquoise arrows) Fam134b isoforms. Primer sequences are listed in Materials and Methods . TFEB (green) subcellular localization analysis in wild type (control) and FGFR3;4 KO chondrocytes treated with FGF18 (50 ng/ml) for 16 h. Torin 1 was used at 1 μM for 2 h as positive control of TFEB nuclear translocation. Nuclei were stained with DAPI (blue). Scale bar 15 μm. Quantification is shown in Fig 3 A. Chromatin immunoprecipitation experiment in TFEB‐WT overexpressing cells treated with FGF18 (50 ng/ml) for 16 h, showing enrichment of TFEB binding on Mucolipin‐1 promoter upon FGF18 treatment. N = 3 biological replicates. Mean ± standard error (sd). Student's unpaired t ‐test ** P

    Techniques Used: Activity Assay, Western Blot, Expressing, Real-time Polymerase Chain Reaction, Positive Control, Translocation Assay, Staining, Chromatin Immunoprecipitation, Binding Assay

    Starvation induces ER‐phagy in RCS and HeLa cells through TFEB and TFE3 Western blot analysis of phospho‐P70S6K (T389), P70S6K, and phospho‐TFEB (S142) in RCS cultured in complete medium or HBSS for 16 h. β‐actin was used as a loading control. Bar graph shows quantification of phospho‐TFEB (S142) normalized to β‐actin. Mean ± standard error of the mean (SEM) of N = 3 biological replicates. Student's paired t ‐test * P
    Figure Legend Snippet: Starvation induces ER‐phagy in RCS and HeLa cells through TFEB and TFE3 Western blot analysis of phospho‐P70S6K (T389), P70S6K, and phospho‐TFEB (S142) in RCS cultured in complete medium or HBSS for 16 h. β‐actin was used as a loading control. Bar graph shows quantification of phospho‐TFEB (S142) normalized to β‐actin. Mean ± standard error of the mean (SEM) of N = 3 biological replicates. Student's paired t ‐test * P

    Techniques Used: Western Blot, Cell Culture

    38) Product Images from "The deubiquitinase USP7 uses a distinct ubiquitin-like domain to deubiquitinate NF-ĸB subunits"

    Article Title: The deubiquitinase USP7 uses a distinct ubiquitin-like domain to deubiquitinate NF-ĸB subunits

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA120.014113

    USP7 inhibition selectively inhibits LPS-induced expression of NF-ĸB target genes. Murine bone marrow–derived macrophages were stimulated with LPS (100 ng/ml) with or without 30 min of pretreatment with the USP7 inhibitor HBX14,108 (USP7i) (10 μ m ), and gene expression was analyzed by microarray. A , hierarchical clustering shows selective inhibition of LPS-induced gene expression by USP7 inhibitor treatment. The heat map displays differentially expressed genes ( p .adj
    Figure Legend Snippet: USP7 inhibition selectively inhibits LPS-induced expression of NF-ĸB target genes. Murine bone marrow–derived macrophages were stimulated with LPS (100 ng/ml) with or without 30 min of pretreatment with the USP7 inhibitor HBX14,108 (USP7i) (10 μ m ), and gene expression was analyzed by microarray. A , hierarchical clustering shows selective inhibition of LPS-induced gene expression by USP7 inhibitor treatment. The heat map displays differentially expressed genes ( p .adj

    Techniques Used: Inhibition, Expressing, Derivative Assay, Microarray

    The Ubl2 domain is required for USP7 deubiquitination of p65 but not RelB. HEK293T cells were transfected with HA-tagged ubiquitin, USP7, USP7ΔUbl2, and p65 ( A ) or RelB ( B ). Denatured whole cell lysates were immunoprecipitated with anti-p65 ( A ) or anti-RelB ( B ) and analyzed by Western blotting ( WB ) with anti-HA antibody and anti-p65 ( A ) or anti-RelB ( B ) antibody. The expression of transfected USP7, USP7ΔUbl2, p65, and RelB in lysates used for immunoprecipitations ( IP , input ) was measured by Western blotting using antibodies against USP7, p65, and RelB. The positions of molecular mass markers are indicated to the right of each Western blot. The data are representative of at least three independent experiments.
    Figure Legend Snippet: The Ubl2 domain is required for USP7 deubiquitination of p65 but not RelB. HEK293T cells were transfected with HA-tagged ubiquitin, USP7, USP7ΔUbl2, and p65 ( A ) or RelB ( B ). Denatured whole cell lysates were immunoprecipitated with anti-p65 ( A ) or anti-RelB ( B ) and analyzed by Western blotting ( WB ) with anti-HA antibody and anti-p65 ( A ) or anti-RelB ( B ) antibody. The expression of transfected USP7, USP7ΔUbl2, p65, and RelB in lysates used for immunoprecipitations ( IP , input ) was measured by Western blotting using antibodies against USP7, p65, and RelB. The positions of molecular mass markers are indicated to the right of each Western blot. The data are representative of at least three independent experiments.

    Techniques Used: Transfection, Immunoprecipitation, Western Blot, Expressing

    Peptide array analysis identifies distinct regions of USP7 Ubl domains with potential for interacting with p65. A , peptide arrays of immobilized overlapping 18-mer peptides, each shifted to the C terminus by 4 amino acids, encompassing the C-terminal region of USP7 were generated. Peptide identifiers and the corresponding amino acids and domain location in USP7 are shown. B , arrays were probed with recombinant GST or GST-p65 and detected by immunoblotting with anti-GST antibody. Positive GST-p65 binding to USP7 peptides is indicated by black spots . The data shown are representative of duplicate arrays. Peptide identifiers shown in A are indicated. C , schematic of the C-terminal regions of USP7 showing the location of peptides that interact with p65 identified in the peptide array.
    Figure Legend Snippet: Peptide array analysis identifies distinct regions of USP7 Ubl domains with potential for interacting with p65. A , peptide arrays of immobilized overlapping 18-mer peptides, each shifted to the C terminus by 4 amino acids, encompassing the C-terminal region of USP7 were generated. Peptide identifiers and the corresponding amino acids and domain location in USP7 are shown. B , arrays were probed with recombinant GST or GST-p65 and detected by immunoblotting with anti-GST antibody. Positive GST-p65 binding to USP7 peptides is indicated by black spots . The data shown are representative of duplicate arrays. Peptide identifiers shown in A are indicated. C , schematic of the C-terminal regions of USP7 showing the location of peptides that interact with p65 identified in the peptide array.

    Techniques Used: Peptide Microarray, Generated, Recombinant, Binding Assay

    The Ubl2 domain of USP7 is required for interaction with c-Rel but not for interaction with p53, EBNA1, DAXX, or RelB. HEK293T cells were transfected with the indicated expression plasmids and whole cell lysates immunoprecipitated ( IP ) with anti-FLAG ( A ), anti-HA ( B ), anti-DAXX ( C ), anti-c-Rel ( D ), or anti-RelB ( E ). The expression of USP7-FLAG, USP7ΔUbl2-FLAG, p53, EBNA1-HA-FLAG, DAXX-FLAG, c-Rel-FLAG, and RelB-FLAG in whole cell lysates used for immunoprecipitations ( input ) was measured by Western blotting ( WB ) analysis with the indicated antibodies. The positions of molecular mass markers are indicated to the right of each Western blot. The data are representative of at least three independent experiments.
    Figure Legend Snippet: The Ubl2 domain of USP7 is required for interaction with c-Rel but not for interaction with p53, EBNA1, DAXX, or RelB. HEK293T cells were transfected with the indicated expression plasmids and whole cell lysates immunoprecipitated ( IP ) with anti-FLAG ( A ), anti-HA ( B ), anti-DAXX ( C ), anti-c-Rel ( D ), or anti-RelB ( E ). The expression of USP7-FLAG, USP7ΔUbl2-FLAG, p53, EBNA1-HA-FLAG, DAXX-FLAG, c-Rel-FLAG, and RelB-FLAG in whole cell lysates used for immunoprecipitations ( input ) was measured by Western blotting ( WB ) analysis with the indicated antibodies. The positions of molecular mass markers are indicated to the right of each Western blot. The data are representative of at least three independent experiments.

    Techniques Used: Transfection, Expressing, Immunoprecipitation, Western Blot

    The Ubl2 domain of USP7 is required for interaction with p65. A , schematic representation of USP7 mutations used to test interaction with p65. USP7 contains an N-terminal MATH domain, a central catalytic domain ( CD ), and five C-terminal Ubl domains. B–F , HEK293T cells were transfected with p65 and the indicated FLAG-tagged USP7 plasmids. USP7 was immunoprecipitated ( IP ) from whole cell lysates using anti-FLAG antibody and analyzed by Western blotting ( WB ) with anti-p65 antibody. The expression of p65 and USP7 in lysates used for immunoprecipitation ( input ) was measured by Western blotting using anti-FLAG and anti-p65 antibodies. Western blotting of inputs with anti–β-actin was used to verify equal protein loading. The positions of molecular mass markers are indicated to the right of each Western blot. The data are representative of at least three independent experiments.
    Figure Legend Snippet: The Ubl2 domain of USP7 is required for interaction with p65. A , schematic representation of USP7 mutations used to test interaction with p65. USP7 contains an N-terminal MATH domain, a central catalytic domain ( CD ), and five C-terminal Ubl domains. B–F , HEK293T cells were transfected with p65 and the indicated FLAG-tagged USP7 plasmids. USP7 was immunoprecipitated ( IP ) from whole cell lysates using anti-FLAG antibody and analyzed by Western blotting ( WB ) with anti-p65 antibody. The expression of p65 and USP7 in lysates used for immunoprecipitation ( input ) was measured by Western blotting using anti-FLAG and anti-p65 antibodies. Western blotting of inputs with anti–β-actin was used to verify equal protein loading. The positions of molecular mass markers are indicated to the right of each Western blot. The data are representative of at least three independent experiments.

    Techniques Used: Transfection, Immunoprecipitation, Western Blot, Expressing

    Identification of amino acids important for USP7 interaction with p65. A , the 18 amino acids of USP7-derived peptides of interest were sequentially substituted with alanine, probed with GST-p65, and detected by immunoblotting with anti-GST antibody. The peptides are labeled according to Fig. 5 . p65 binding was quantified and calculated as a percentage binding of the parent unsubstituted peptide (-) on the same array. Alanine substitutions that resulted in less than 60% binding are indicated by asterisks . The corresponding USP7 amino acid number for first residue of each peptide is shown above each individual substitution peptide series. B , the structure of each Ubl domain with amino acids identified by alanine-scanning experiments indicated in red . The images were generated using Protein Data Bank structure 2YLM . C , HEK293T cells were transfected with p65 and FLAG-tagged USP7, USP7 mutated at residues 757–760, and USP7 lacking the Ubl2 domain (USP7ΔUbl2). p65 was immunoprecipitated ( IP ) from whole cell lysates using anti-p65 antibody and analyzed by Western blotting ( WB ) with anti-FLAG antibody. The expression of p65 and USP7 in lysates used for immunoprecipitation ( input ) was measured by Western blotting using anti-FLAG and anti-p65 antibodies. The positions of molecular mass markers are indicated to the right of each Western blot.
    Figure Legend Snippet: Identification of amino acids important for USP7 interaction with p65. A , the 18 amino acids of USP7-derived peptides of interest were sequentially substituted with alanine, probed with GST-p65, and detected by immunoblotting with anti-GST antibody. The peptides are labeled according to Fig. 5 . p65 binding was quantified and calculated as a percentage binding of the parent unsubstituted peptide (-) on the same array. Alanine substitutions that resulted in less than 60% binding are indicated by asterisks . The corresponding USP7 amino acid number for first residue of each peptide is shown above each individual substitution peptide series. B , the structure of each Ubl domain with amino acids identified by alanine-scanning experiments indicated in red . The images were generated using Protein Data Bank structure 2YLM . C , HEK293T cells were transfected with p65 and FLAG-tagged USP7, USP7 mutated at residues 757–760, and USP7 lacking the Ubl2 domain (USP7ΔUbl2). p65 was immunoprecipitated ( IP ) from whole cell lysates using anti-p65 antibody and analyzed by Western blotting ( WB ) with anti-FLAG antibody. The expression of p65 and USP7 in lysates used for immunoprecipitation ( input ) was measured by Western blotting using anti-FLAG and anti-p65 antibodies. The positions of molecular mass markers are indicated to the right of each Western blot.

    Techniques Used: Derivative Assay, Labeling, Binding Assay, Generated, Transfection, Immunoprecipitation, Western Blot, Expressing

    39) Product Images from "Mediator Kinase Inhibition Further Activates Super-Enhancer Associated Genes in AML"

    Article Title: Mediator Kinase Inhibition Further Activates Super-Enhancer Associated Genes in AML

    Journal: Nature

    doi: 10.1038/nature14904

    CA inhibition of and binding to CDK8 ( a ) CA inhibition of CDK8 module phosphorylation of CDK8 and STAT1 S727 substrate (mean ± s.e.m., n =3 biological replicates, one of two experiments shown, autorad in Supplementary Figure 1 ). ( b ) CA inhibition in vitro of CDK8 module activity but not CDK12:Cyclin K or CDK13:Cyclin K activity up to 10 μM. Equal amounts (silver stain) of GST-CTD were used as the substrate in in vitro kinase assays. The amount of each kinase used was empirically determined to give approximately the same GST-CTD signal under the assay conditions. “ns” is no substrate (kinase only) and “GST-CTD-P” is phosphorylated GST-CTD. One of four experiments shown. ( c ) Immunoblot showing that CA selectively and dose-dependently inhibits capture of native CDK8 (IC 50 ~10 nM) and CDK19 (IC 50 ~ 100 nM) from MOLM-14 lysates but did not inhibit capture of CDK9, CDK12, CDK13, ROCK1, ROCK2 or GSG2. One of two experiments shown, full scan in Supplementary Figure 1 . ( d ) Immunoblots showing CA inhibition of CDK8-dependent IFN-γ-stimulated STAT1 S727 phosphorylation in MOLM-14 cells and CA inhibition of TGF-β-stimulated Smad2 T220 and Smad3 T179 phosphorylation in HaCaT cells (IC 50
    Figure Legend Snippet: CA inhibition of and binding to CDK8 ( a ) CA inhibition of CDK8 module phosphorylation of CDK8 and STAT1 S727 substrate (mean ± s.e.m., n =3 biological replicates, one of two experiments shown, autorad in Supplementary Figure 1 ). ( b ) CA inhibition in vitro of CDK8 module activity but not CDK12:Cyclin K or CDK13:Cyclin K activity up to 10 μM. Equal amounts (silver stain) of GST-CTD were used as the substrate in in vitro kinase assays. The amount of each kinase used was empirically determined to give approximately the same GST-CTD signal under the assay conditions. “ns” is no substrate (kinase only) and “GST-CTD-P” is phosphorylated GST-CTD. One of four experiments shown. ( c ) Immunoblot showing that CA selectively and dose-dependently inhibits capture of native CDK8 (IC 50 ~10 nM) and CDK19 (IC 50 ~ 100 nM) from MOLM-14 lysates but did not inhibit capture of CDK9, CDK12, CDK13, ROCK1, ROCK2 or GSG2. One of two experiments shown, full scan in Supplementary Figure 1 . ( d ) Immunoblots showing CA inhibition of CDK8-dependent IFN-γ-stimulated STAT1 S727 phosphorylation in MOLM-14 cells and CA inhibition of TGF-β-stimulated Smad2 T220 and Smad3 T179 phosphorylation in HaCaT cells (IC 50

    Techniques Used: Inhibition, Binding Assay, In Vitro, Activity Assay, Silver Staining, Western Blot

    Mediator kinases mediate the antiproliferative activity of CA ( a ) We evaluated point mutations to CDK8 residues lining the CA binding pocket: Ala155, His106, Asp103, and Trp105. Expression of CDK8 A155I, A155F, A155Q, H106K and D103E in MOLM-14 cells afforded only modest desensitization to CA. Differential sensitivity of MOLM-14 cells to CA upon expression of indicated mutant FLAG-CDK8 proteins (mean ± s.e.m., n =3 biological replicates, experiment performed once). ( b ) Immunoblots showing that FLAG-CDK8 or FLAG-CDK19 and FLAG-CDK8 W105M or FLAG-CDK19 W105M are expressed at similar levels in MOLM-14, MV4;11, and SKNO-1 cells (experiment performed once, full scan in Supplementary Figure 1 ). ( c ) Differential sensitivity of MV4;11 and SKNO-1 cells to CA upon expression of FLAG-CDK8, FLAG-CDK19, FLAG-CDK8 W105M and FLAG-CDK19 W105M, legend as in d (mean ± s.e.m., n =3 biological replicates, one of two experiments shown). ( d ) Control showing that expression of FLAG-CDK8 W105M or FLAG-CDK19 W105M in MOLM-14, MV4;11, and SKNO-1 cells does not confer resistance to antiproliferative agents paclitaxel and doxorubicin (mean ± s.e.m., n =3 biological replicates, one of two experiments shown). ( e ) Purified FLAG-CDK8 W105M and FLAG-CDK19 W105M remain catalytically active for phosphorylation of CTD in vitro but are resistant to inhibition by CA (mean ± s.e.m., n =3 biological replicates, experiment performed once). ( f ) Representative autorad and silver stain images supporting quantitation shown in ( e ). ( g ) Sequence alignment of human CDKs. Sequence alignment was performed on segments of CDK1-20 using Clustal Omega. The unique Trp105 residue in CDK8 and CDK19 is highlighted in red, and is absent from other CDKs (orange box). UniProt Knowledgebase entries: CDK1, P06493; CDK2, P24941; CDK3, Q00526; CDK4, P11802; CDK5, Q00535; CDK6, Q00534; CDK7, P50613; CDK8, P49336; CDK9, P50750; CDK10, Q15131; CDK11A, Q9UQ88; CDK11B, P21127; CDK12, Q9NYV4; CDK13, Q14004; CDK14, O94921; CDK15, Q96Q40; CDK16, Q00536; CDK17, Q00537; CDK18, Q07002; CDK19, Q9BWU1; CDK20, Q8IZL9.
    Figure Legend Snippet: Mediator kinases mediate the antiproliferative activity of CA ( a ) We evaluated point mutations to CDK8 residues lining the CA binding pocket: Ala155, His106, Asp103, and Trp105. Expression of CDK8 A155I, A155F, A155Q, H106K and D103E in MOLM-14 cells afforded only modest desensitization to CA. Differential sensitivity of MOLM-14 cells to CA upon expression of indicated mutant FLAG-CDK8 proteins (mean ± s.e.m., n =3 biological replicates, experiment performed once). ( b ) Immunoblots showing that FLAG-CDK8 or FLAG-CDK19 and FLAG-CDK8 W105M or FLAG-CDK19 W105M are expressed at similar levels in MOLM-14, MV4;11, and SKNO-1 cells (experiment performed once, full scan in Supplementary Figure 1 ). ( c ) Differential sensitivity of MV4;11 and SKNO-1 cells to CA upon expression of FLAG-CDK8, FLAG-CDK19, FLAG-CDK8 W105M and FLAG-CDK19 W105M, legend as in d (mean ± s.e.m., n =3 biological replicates, one of two experiments shown). ( d ) Control showing that expression of FLAG-CDK8 W105M or FLAG-CDK19 W105M in MOLM-14, MV4;11, and SKNO-1 cells does not confer resistance to antiproliferative agents paclitaxel and doxorubicin (mean ± s.e.m., n =3 biological replicates, one of two experiments shown). ( e ) Purified FLAG-CDK8 W105M and FLAG-CDK19 W105M remain catalytically active for phosphorylation of CTD in vitro but are resistant to inhibition by CA (mean ± s.e.m., n =3 biological replicates, experiment performed once). ( f ) Representative autorad and silver stain images supporting quantitation shown in ( e ). ( g ) Sequence alignment of human CDKs. Sequence alignment was performed on segments of CDK1-20 using Clustal Omega. The unique Trp105 residue in CDK8 and CDK19 is highlighted in red, and is absent from other CDKs (orange box). UniProt Knowledgebase entries: CDK1, P06493; CDK2, P24941; CDK3, Q00526; CDK4, P11802; CDK5, Q00535; CDK6, Q00534; CDK7, P50613; CDK8, P49336; CDK9, P50750; CDK10, Q15131; CDK11A, Q9UQ88; CDK11B, P21127; CDK12, Q9NYV4; CDK13, Q14004; CDK14, O94921; CDK15, Q96Q40; CDK16, Q00536; CDK17, Q00537; CDK18, Q07002; CDK19, Q9BWU1; CDK20, Q8IZL9.

    Techniques Used: Activity Assay, Binding Assay, Expressing, Mutagenesis, Western Blot, Purification, In Vitro, Inhibition, Silver Staining, Quantitation Assay, Sequencing

    40) Product Images from "SUMO Ligase Protein Inhibitor of Activated STAT1 (PIAS1) Is a Constituent Promyelocytic Leukemia Nuclear Body Protein That Contributes to the Intrinsic Antiviral Immune Response to Herpes Simplex Virus 1"

    Article Title: SUMO Ligase Protein Inhibitor of Activated STAT1 (PIAS1) Is a Constituent Promyelocytic Leukemia Nuclear Body Protein That Contributes to the Intrinsic Antiviral Immune Response to Herpes Simplex Virus 1

    Journal: Journal of Virology

    doi: 10.1128/JVI.00426-16

    Ectopic PIAS1 associates with and disrupts PML-NBs in a SIM- and SUMO ligase-dependent manner. (A) Diagram highlighting the PIAS family conserved functional domains (gray) within PIAS1. SAP, SAF-A/B, Acinus, and PIAS; PINIT, “PINIT” motif; SP-RING, Siz/PIAS-RING zinc finger; SIM, SUMO interaction motif; C-Term, variable C-terminal domain. (B and C) Confocal microscopy images show the nuclear localization of eYFP (B) or eYFP-PIAS1 (C) with respect to PML. EYFP or eYFP-PIAS1 expression was DOX induced (+Dox), or not (−Dox), for 8 or 16 h. PML (red) was visualized by indirect immunofluorescence. (D and E) Emission spectra show the pixel intensity and colocalization between PML and eYFP (D) or eYFP-PIAS1 (E) in the regions indicated by white bars in panel B or C, respectively. (F) Confocal microscopy images show the nuclear localization of eYFP or catalytically inactive eYFP-PIAS1, with (eYFP.P1.C351A.mSIM) or without (eYFP.P1.C351A) a mutant SIM, with respect to PML-NBs. Expression of eYFP or eYFP-PIAS1 mutant proteins was DOX induced for 16 h. PML-NBs were identified by the accumulation of PML. PIAS1 (pAb; red) and PML (cyan) were visualized by indirect immunofluorescence. Nuclei were stained with DAPI (blue). pAb, polyclonal antibody.
    Figure Legend Snippet: Ectopic PIAS1 associates with and disrupts PML-NBs in a SIM- and SUMO ligase-dependent manner. (A) Diagram highlighting the PIAS family conserved functional domains (gray) within PIAS1. SAP, SAF-A/B, Acinus, and PIAS; PINIT, “PINIT” motif; SP-RING, Siz/PIAS-RING zinc finger; SIM, SUMO interaction motif; C-Term, variable C-terminal domain. (B and C) Confocal microscopy images show the nuclear localization of eYFP (B) or eYFP-PIAS1 (C) with respect to PML. EYFP or eYFP-PIAS1 expression was DOX induced (+Dox), or not (−Dox), for 8 or 16 h. PML (red) was visualized by indirect immunofluorescence. (D and E) Emission spectra show the pixel intensity and colocalization between PML and eYFP (D) or eYFP-PIAS1 (E) in the regions indicated by white bars in panel B or C, respectively. (F) Confocal microscopy images show the nuclear localization of eYFP or catalytically inactive eYFP-PIAS1, with (eYFP.P1.C351A.mSIM) or without (eYFP.P1.C351A) a mutant SIM, with respect to PML-NBs. Expression of eYFP or eYFP-PIAS1 mutant proteins was DOX induced for 16 h. PML-NBs were identified by the accumulation of PML. PIAS1 (pAb; red) and PML (cyan) were visualized by indirect immunofluorescence. Nuclei were stained with DAPI (blue). pAb, polyclonal antibody.

    Techniques Used: Functional Assay, Confocal Microscopy, Expressing, Immunofluorescence, Mutagenesis, Staining

    PIAS1 localization at PML-NBs requires SUMO-modified PML. (A) Western blots show PML and PIAS1 protein levels in transgenic cells that express PML-specific shRNA (shPML) or a nontargeted control sequence (shCtrl). Whole-cell lysates were resolved by Tris-glycine SDS-PAGE. Membranes were probed for PML or PIAS1 and for actin as a loading control. Molecular masses are indicated. (B) Confocal microscopy images show the nuclear localization of PIAS1 in transgenic HFt cells that express shPML or shCtrl. PIAS1 (red) and PML (green) were detected by indirect immunofluorescence. (C and D) Confocal microscopy images show PIAS1 localization with respect to PML isoform I in PML-depleted transgenic HFt cells stably reconstituted with PML.I (eYFP.PML.I [C]) or SUMOylation-deficient PML.I (eYFP.PML.I.4KR [D]). PIAS1 (red), Daxx (red), Sp100 (red), SUMO1 (red), SUMO2/3 (red), and PML (cyan) were detected by indirect immunofluorescence. Nuclei were stained with DAPI (blue). mAb, monoclonal antibody.
    Figure Legend Snippet: PIAS1 localization at PML-NBs requires SUMO-modified PML. (A) Western blots show PML and PIAS1 protein levels in transgenic cells that express PML-specific shRNA (shPML) or a nontargeted control sequence (shCtrl). Whole-cell lysates were resolved by Tris-glycine SDS-PAGE. Membranes were probed for PML or PIAS1 and for actin as a loading control. Molecular masses are indicated. (B) Confocal microscopy images show the nuclear localization of PIAS1 in transgenic HFt cells that express shPML or shCtrl. PIAS1 (red) and PML (green) were detected by indirect immunofluorescence. (C and D) Confocal microscopy images show PIAS1 localization with respect to PML isoform I in PML-depleted transgenic HFt cells stably reconstituted with PML.I (eYFP.PML.I [C]) or SUMOylation-deficient PML.I (eYFP.PML.I.4KR [D]). PIAS1 (red), Daxx (red), Sp100 (red), SUMO1 (red), SUMO2/3 (red), and PML (cyan) were detected by indirect immunofluorescence. Nuclei were stained with DAPI (blue). mAb, monoclonal antibody.

    Techniques Used: Modification, Western Blot, Transgenic Assay, shRNA, Sequencing, SDS Page, Confocal Microscopy, Immunofluorescence, Stable Transfection, Staining

    PIAS1 and PIAS4 cooperatively restrict ICP0-null mutant HSV-1 replication. (A and B) Confocal microscopy images show the localization of PIAS1 and PIAS4 in HSV-1-infected cells. HFt cells were infected with 2 or 0.002 PFU per cell of ICP0-null mutant (A) or wild-type HSV-1 (B), respectively, for 16 h. PIAS1 (red), PIAS4 (red), and PML (cyan) were visualized by indirect immunofluorescence. Replication compartments were identified by the accumulation of eYFP.ICP4. Insets (dashed boxes in panel A) highlight a region where PIAS1 and PML colocalize within a replication compartment. Nuclei were stained with DAPI (blue). (C) Western blots show PIAS1 or PIAS4 protein levels in transgenic HFt cells that express shRNA against PIAS1 (shPIAS1), PIAS4 (shPIAS4), or a nontargeted control (shCtrl). Whole-cell lysates were resolved by Tris-glycine SDS-PAGE. Membranes were probed for PIAS1 or PIAS4 and for actin as a loading control. Molecular masses are indicated. Puro, puromycin. (D) Bar graph showing the average relative PFE of wild-type or ICP0-null mutant HSV-1 in transgenic HFt cells that express shPIAS1, shPIAS4, or shCtrl. The number of plaques for each strain is expressed relative to the corresponding number of plaques for that strain in cells that express shCtrl. Means and SD are shown ( n = 6).
    Figure Legend Snippet: PIAS1 and PIAS4 cooperatively restrict ICP0-null mutant HSV-1 replication. (A and B) Confocal microscopy images show the localization of PIAS1 and PIAS4 in HSV-1-infected cells. HFt cells were infected with 2 or 0.002 PFU per cell of ICP0-null mutant (A) or wild-type HSV-1 (B), respectively, for 16 h. PIAS1 (red), PIAS4 (red), and PML (cyan) were visualized by indirect immunofluorescence. Replication compartments were identified by the accumulation of eYFP.ICP4. Insets (dashed boxes in panel A) highlight a region where PIAS1 and PML colocalize within a replication compartment. Nuclei were stained with DAPI (blue). (C) Western blots show PIAS1 or PIAS4 protein levels in transgenic HFt cells that express shRNA against PIAS1 (shPIAS1), PIAS4 (shPIAS4), or a nontargeted control (shCtrl). Whole-cell lysates were resolved by Tris-glycine SDS-PAGE. Membranes were probed for PIAS1 or PIAS4 and for actin as a loading control. Molecular masses are indicated. Puro, puromycin. (D) Bar graph showing the average relative PFE of wild-type or ICP0-null mutant HSV-1 in transgenic HFt cells that express shPIAS1, shPIAS4, or shCtrl. The number of plaques for each strain is expressed relative to the corresponding number of plaques for that strain in cells that express shCtrl. Means and SD are shown ( n = 6).

    Techniques Used: Mutagenesis, Confocal Microscopy, Infection, Immunofluorescence, Staining, Western Blot, Transgenic Assay, shRNA, SDS Page

    PIAS1 is not essential for PML-NB formation or PML SUMOylation but does enhance the accumulation of SUMO1 in domains that contain infecting HSV-1 genomes. (A) Western blots show PIAS1 protein levels in transgenic HFt cells that express PIAS1-specific (shPIAS1) or nontargeted control (shCtrl) shRNA. Whole-cell lysates were resolved by Tris-glycine SDS-PAGE. Membranes were probed for PIAS1 or PML and for actin as a loading control. Molecular masses are indicated. (B) Confocal microscopy images show the localization of PML in transgenic shPIAS1- or shCtrl-expressing cells. PML (green) and PIAS1 (red) were detected by indirect immunofluorescence. (C) Confocal microscopy images show the localization of Daxx, SUMO1, or SUMO2/3 in mock-infected HFt cells that express shPIAS1 or shCtrl. Daxx (green), PIAS1 (red), SUMO1 (cyan), and SUMO2/3 (cyan) were detected by indirect immunofluorescence. (D) Confocal microscopy images show the nuclear localization of PML, Daxx, SUMO1, or SUMO2/3 during ICP0-null mutant HSV-1 infection of transgenic HFt cells that express shPIAS1 or shCtrl. Cells were infected with 2 PFU of ICP0-null mutant HSV-1 per cell for 16 h. PIAS1 (red), PML (cyan), Daxx (cyan), SUMO1 (cyan), and SUMO2/3 (cyan) were detected by indirect immunofluorescence. The nuclear edge associated with HSV-1 genome entry was identified by eYFP.ICP4 localization. Nuclei were stained with DAPI (blue).
    Figure Legend Snippet: PIAS1 is not essential for PML-NB formation or PML SUMOylation but does enhance the accumulation of SUMO1 in domains that contain infecting HSV-1 genomes. (A) Western blots show PIAS1 protein levels in transgenic HFt cells that express PIAS1-specific (shPIAS1) or nontargeted control (shCtrl) shRNA. Whole-cell lysates were resolved by Tris-glycine SDS-PAGE. Membranes were probed for PIAS1 or PML and for actin as a loading control. Molecular masses are indicated. (B) Confocal microscopy images show the localization of PML in transgenic shPIAS1- or shCtrl-expressing cells. PML (green) and PIAS1 (red) were detected by indirect immunofluorescence. (C) Confocal microscopy images show the localization of Daxx, SUMO1, or SUMO2/3 in mock-infected HFt cells that express shPIAS1 or shCtrl. Daxx (green), PIAS1 (red), SUMO1 (cyan), and SUMO2/3 (cyan) were detected by indirect immunofluorescence. (D) Confocal microscopy images show the nuclear localization of PML, Daxx, SUMO1, or SUMO2/3 during ICP0-null mutant HSV-1 infection of transgenic HFt cells that express shPIAS1 or shCtrl. Cells were infected with 2 PFU of ICP0-null mutant HSV-1 per cell for 16 h. PIAS1 (red), PML (cyan), Daxx (cyan), SUMO1 (cyan), and SUMO2/3 (cyan) were detected by indirect immunofluorescence. The nuclear edge associated with HSV-1 genome entry was identified by eYFP.ICP4 localization. Nuclei were stained with DAPI (blue).

    Techniques Used: Western Blot, Transgenic Assay, shRNA, SDS Page, Confocal Microscopy, Expressing, Immunofluorescence, Infection, Mutagenesis, Staining

    ICP0 disperses PIAS1 away from domains that contain infecting HSV-1 genomes without targeting it for proteasomal degradation. (A) Western blots show PIAS1 protein levels during wild-type or ICP0-null mutant HSV-1 infection. HFt cells were infected with 10 PFU of wild-type (HSV-1) or ICP0-null mutant (ΔICP0) HSV-1 per cell in the presence (+) or absence (−) of the proteasome inhibitor MG132. Whole-cell lysates were harvested at 3, 6, or 9 h postinfection (hpi), and proteins were resolved by Tris-Tricine SDS-PAGE. Membranes were probed for PIAS1, PML as an example of an ICP0 substrate, ICP0, ICP4, and UL42 to show the progression of infection, and actin as a loading control. Molecular masses are indicated. (B) Bar graph shows the average relative levels of PIAS1 during infection with wild-type or ICP0-null mutant HSV-1. The intensities of PIAS1 protein bands were quantitated from Western blots as in panel A, normalized to their respective loading control, and presented as a ratio to the level in mock-infected cells at 9 hpi (1.0). Means and standard error of the means (SEM) are shown ( n = 7). *, P
    Figure Legend Snippet: ICP0 disperses PIAS1 away from domains that contain infecting HSV-1 genomes without targeting it for proteasomal degradation. (A) Western blots show PIAS1 protein levels during wild-type or ICP0-null mutant HSV-1 infection. HFt cells were infected with 10 PFU of wild-type (HSV-1) or ICP0-null mutant (ΔICP0) HSV-1 per cell in the presence (+) or absence (−) of the proteasome inhibitor MG132. Whole-cell lysates were harvested at 3, 6, or 9 h postinfection (hpi), and proteins were resolved by Tris-Tricine SDS-PAGE. Membranes were probed for PIAS1, PML as an example of an ICP0 substrate, ICP0, ICP4, and UL42 to show the progression of infection, and actin as a loading control. Molecular masses are indicated. (B) Bar graph shows the average relative levels of PIAS1 during infection with wild-type or ICP0-null mutant HSV-1. The intensities of PIAS1 protein bands were quantitated from Western blots as in panel A, normalized to their respective loading control, and presented as a ratio to the level in mock-infected cells at 9 hpi (1.0). Means and standard error of the means (SEM) are shown ( n = 7). *, P

    Techniques Used: Western Blot, Mutagenesis, Infection, SDS Page

    PIAS1 is a PML-NB constituent protein. (A) Confocal microscopy images show the nuclear localization of PIAS1-4 with respect to PML, the major PML-NB scaffolding protein, in HFt cells. The localization of Daxx, a major PML-NB constituent protein, with respect to PML is shown for comparison. PIAS1-4 (red), Daxx (red), and PML (green) were visualized by indirect immunofluorescence. Cut mask (yellow) highlights regions of colocalization; weighted colocalization coefficients are indicated. (B) Box-and-whisker plot showing the distribution of individual weighted colocalization coefficients of Daxx or PIAS1 to -4 with PML. The number of nuclei evaluated for each pairwise comparison is shown. Boxes, upper to lower quartiles; diamonds, means; horizontal lines, medians; upper whiskers, maximum values; lower whiskers, minimum values. (C) Confocal microscopy images show the nuclear localization of PIAS1 and PML in HFs, HEL, HepaRG, or RPE cells. PIAS1 (red) and PML (green) were visualized by indirect immunofluorescence. Nuclei were stained with DAPI (blue).
    Figure Legend Snippet: PIAS1 is a PML-NB constituent protein. (A) Confocal microscopy images show the nuclear localization of PIAS1-4 with respect to PML, the major PML-NB scaffolding protein, in HFt cells. The localization of Daxx, a major PML-NB constituent protein, with respect to PML is shown for comparison. PIAS1-4 (red), Daxx (red), and PML (green) were visualized by indirect immunofluorescence. Cut mask (yellow) highlights regions of colocalization; weighted colocalization coefficients are indicated. (B) Box-and-whisker plot showing the distribution of individual weighted colocalization coefficients of Daxx or PIAS1 to -4 with PML. The number of nuclei evaluated for each pairwise comparison is shown. Boxes, upper to lower quartiles; diamonds, means; horizontal lines, medians; upper whiskers, maximum values; lower whiskers, minimum values. (C) Confocal microscopy images show the nuclear localization of PIAS1 and PML in HFs, HEL, HepaRG, or RPE cells. PIAS1 (red) and PML (green) were visualized by indirect immunofluorescence. Nuclei were stained with DAPI (blue).

    Techniques Used: Confocal Microscopy, Scaffolding, Immunofluorescence, Whisker Assay, Staining

    PIAS1 repression of ICP0-null mutant HSV-1 replication is additive to that of PML. (A) Bar graph showing the average relative levels of PML or PIAS1 mRNA in transgenic HFt cells that express shRNA against PML (shPML), PIAS1 (shPIAS1), or a nontargeted control (shCtrl). PML or PIAS1 mRNA levels were determined using the TaqMan system of quantitative RT-PCR. Values normalized to GAPDH expression using the threshold cycle (ΔΔ C T ) method are expressed relative to mock-infected cells (1.0). Means and standard deviations (SD) are shown ( n > 3). (B) Western blots show PML or PIAS1 protein levels in transgenic HFt cells that express shPML, shPIAS1, or shCtrl. Whole-cell lysates were resolved by Tris-glycine SDS-PAGE. Membranes were probed for PML or PIAS1 and for actin as a loading control. Molecular masses are indicated. (C) Bar graph showing the average relative plaque forming efficiency (PFE) of wild-type (HSV-1) or ICP0-null mutant (ΔICP0) HSV-1 in transgenic HFt cells that express shCtrl, shPML, or shPIAS1. The number of plaques for each strain is expressed relative to the corresponding number of plaques for that strain in shCtrl-expressing cells. Means and SD are shown ( n = 6). Neo, neomycin; Puro, puromycin.
    Figure Legend Snippet: PIAS1 repression of ICP0-null mutant HSV-1 replication is additive to that of PML. (A) Bar graph showing the average relative levels of PML or PIAS1 mRNA in transgenic HFt cells that express shRNA against PML (shPML), PIAS1 (shPIAS1), or a nontargeted control (shCtrl). PML or PIAS1 mRNA levels were determined using the TaqMan system of quantitative RT-PCR. Values normalized to GAPDH expression using the threshold cycle (ΔΔ C T ) method are expressed relative to mock-infected cells (1.0). Means and standard deviations (SD) are shown ( n > 3). (B) Western blots show PML or PIAS1 protein levels in transgenic HFt cells that express shPML, shPIAS1, or shCtrl. Whole-cell lysates were resolved by Tris-glycine SDS-PAGE. Membranes were probed for PML or PIAS1 and for actin as a loading control. Molecular masses are indicated. (C) Bar graph showing the average relative plaque forming efficiency (PFE) of wild-type (HSV-1) or ICP0-null mutant (ΔICP0) HSV-1 in transgenic HFt cells that express shCtrl, shPML, or shPIAS1. The number of plaques for each strain is expressed relative to the corresponding number of plaques for that strain in shCtrl-expressing cells. Means and SD are shown ( n = 6). Neo, neomycin; Puro, puromycin.

    Techniques Used: Mutagenesis, Transgenic Assay, shRNA, Quantitative RT-PCR, Expressing, Infection, Western Blot, SDS Page

    The SIM-dependent localization of PIAS1 at domains that contain infecting HSV-1 genomes is enhanced by PML. (A) Confocal microscopy images show PIAS1 localization, or lack of it, at nuclear domains that contain infecting HSV-1 genomes in transgenic PML depleted (shPML) or not (shCtrl) HFt cells within a developing plaque. Cells were infected with 2 PFU of ICP0-null mutant HSV-1 (ΔICP0) per cell for 16 h. PIAS1 (red), PML (cyan), and Daxx (cyan) were detected by indirect immunofluorescence. (B) Confocal microscopy images show the nuclear localization of eYFP or eYFP-PIAS1 mutant proteins in HSV-1-infected cells within a developing plaque. EYFP or eYFP-PIAS1 mutant protein expression was DOX induced for 4 h prior to infection with 2 PFU of ICP0-null mutant HSV-1 (ΔICP0) or 0.002 PFU of wild-type HSV-1 (HSV-1) per cell for 16 h. ICP4 (red) and PML (cyan) were detected by indirect immunofluorescence. The nuclear edge associated with HSV-1 genome entry is identified by ICP4 localization. Nuclei were stained with DAPI (blue). (C) Western blots show the expression level of eYFP-PIAS1 mutant proteins. Protein expression was DOX induced for 0, 8, or 24 h prior to the collection of whole-cell lysates. Membranes were probed for PIAS1 and actin as a loading control. Molecular masses are indicated.
    Figure Legend Snippet: The SIM-dependent localization of PIAS1 at domains that contain infecting HSV-1 genomes is enhanced by PML. (A) Confocal microscopy images show PIAS1 localization, or lack of it, at nuclear domains that contain infecting HSV-1 genomes in transgenic PML depleted (shPML) or not (shCtrl) HFt cells within a developing plaque. Cells were infected with 2 PFU of ICP0-null mutant HSV-1 (ΔICP0) per cell for 16 h. PIAS1 (red), PML (cyan), and Daxx (cyan) were detected by indirect immunofluorescence. (B) Confocal microscopy images show the nuclear localization of eYFP or eYFP-PIAS1 mutant proteins in HSV-1-infected cells within a developing plaque. EYFP or eYFP-PIAS1 mutant protein expression was DOX induced for 4 h prior to infection with 2 PFU of ICP0-null mutant HSV-1 (ΔICP0) or 0.002 PFU of wild-type HSV-1 (HSV-1) per cell for 16 h. ICP4 (red) and PML (cyan) were detected by indirect immunofluorescence. The nuclear edge associated with HSV-1 genome entry is identified by ICP4 localization. Nuclei were stained with DAPI (blue). (C) Western blots show the expression level of eYFP-PIAS1 mutant proteins. Protein expression was DOX induced for 0, 8, or 24 h prior to the collection of whole-cell lysates. Membranes were probed for PIAS1 and actin as a loading control. Molecular masses are indicated.

    Techniques Used: Confocal Microscopy, Transgenic Assay, Infection, Mutagenesis, Immunofluorescence, Expressing, Staining, Western Blot

    Related Articles

    Immunoprecipitation:

    Article Title: Oncogenic exon 2 mutations in Mediator subunit MED12 disrupt allosteric activation of cyclin C-CDK8/19
    Article Snippet: .. Antibodies used for immunoprecipitation and immunoblotting assays were as follows: anti-MED12, Bethyl A300–774A; anti-MED13, Bethyl A301-278A; anti-CDK8, Santa Cruz sc-1521; anti-CDK19, Sigma HPA007053; anti-CycC, BD Pharmingen 558903; anti-GST, GE Healthcare 27-457701. .. Rabbit polyclonal antibodies specific for MED30 and MED4 antibodies are generated as previously described ( ).

    Incubation:

    Article Title: Regulation of cardiac transcription by thyroid hormone and Med13
    Article Snippet: .. Blots were blocked for 1 h at room temperature in 3% Bovine Serum Albumin/1% Polyvinylpyrrolidone and incubated overnight at 4°C with primary antibodies to detect Med12 (Cell Signaling cat#4529, 1:1000), Med13 (Bethyl cat#A301–277A, 1:500), CDK8 (Santa Cruz cat#sc-1521, 1:1000), Med1 (Bethyl cat#A300–793A, 1:1000), and GAPDH (Cell signaling cat#2118, 1:3000). .. Blots were incubated for 2 h with HRP-conjugated secondary antibody and developed with ECL reagents (GE healthcare) using chemiluminescent-sensitive X-ray film (Fisher Scientific).

    other:

    Article Title: The Human CDK8 Subcomplex Is a Histone Kinase That Requires Med12 for Activity and Can Function Independently of Mediator ▿
    Article Snippet: Med13 (A301-278A) and Med23 (A300-425A) were from Bethyl, Inc. Glu tag (ab1267) and His tag (ab9108) were from Abcam.

    Blocking Assay:

    Article Title: De novo mutations in MED13, a component of the Mediator complex, are associated with a novel neurodevelopmental disorder
    Article Snippet: .. Blots were blocked for 1 h at room temperature in LICOR blocking buffer (LICOR #927-40000), then blots were probed (with washes in PBS-T (0.05% Tween-20) and a secondary probe for 1 h after each primary probe) with 1:250 rabbit anti-MED13 (Bethyl #A301-277A) for 3 days at 4 °C, 1:1,000 mouse anti-HDAC2 (clone 3F3, SCBT #sc-81599) overnight at 4 °C as a loading control, and 1:1000 rabbit anti-HSP90 (abcam #ab115660) overnight at 4 °C as an additional loading control. .. Three other primary antibodies were tested for MED13, but did not show sufficient signal to detect MED13 in blood despite detecting MED13 in neural precursor nuclear lysates: Bethyl #278A, Abcam #ab49468, and Abcam #ab76923 (data not shown).

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 90
    Bethyl anti actin ab
    Anti Actin Ab, supplied by Bethyl, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti actin ab/product/Bethyl
    Average 90 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti actin ab - by Bioz Stars, 2020-09
    90/100 stars
      Buy from Supplier

    Image Search Results