pcdna3 1 Thermo Fisher Search Results


  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 79
    Thermo Fisher pcdna3 1 pc3 1 expression vector
    Pcdna3 1 Pc3 1 Expression Vector, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 79/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcdna3 1 pc3 1 expression vector/product/Thermo Fisher
    Average 79 stars, based on 5 article reviews
    Price from $9.99 to $1999.99
    pcdna3 1 pc3 1 expression vector - by Bioz Stars, 2020-02
    79/100 stars
      Buy from Supplier

    99
    Thermo Fisher pcdna3 1
    Fig. 4. γ 1 –σ 1 AP-1 subcomplex is retained on rabaptin-5α beads. ( A ) GST or GST–rabaptin-5α(301–592) were incubated with glutathione–Sepharose and a HeLa cell extract. The western blot of bound (B) and non-bound (NB) fractions was probed with antibodies against γ 1 , β 1/2 , σ 1 , µ 1 and clathrin. ( B ) IF of clathrin and rabaptin-5α in cells transfected with rabaptin-5α or co-transfected with rabaptin-5α and γ 1 -adaptin. Cells were permeabilized, fixed and labelled with a rabbit antibody against rabaptin-5α (green) and X-22 against clathrin (red). ( C ) HeLa cells were labelled with [ 35 S]methionine and the lysate was incubated with GST–rabaptin-5α(301–592). Bound material was eluted with glutathione. Eluate and cell lysate were incubated with antibodies against γ 1 (100/3), β 1/2 (100/1), µ 1 and σ 1 , and protein A beads. Immunoprecipitates were resolved by SDS–PAGE and analysed by phosphorimaging. To detect σ 1 in the GSH eluate, gels were exposed 10 times longer than for γ 1 -adaptin (σ1 contains four times less methionine residues than γ 1 -adaptin). ( D ) Rabaptin-5α and γ 1 -adaptin co-localize on endosomes in the absence of intact AP-1. µ 1 A–/– and µ 1 A+/+ fibroblasts were transfected with <t>rabaptin-5α-pcDNA3.1His.</t> For γ 1 -adaptin labelling in µ 1 A+/+ fibroblasts, cells were extracted with saponin before fixation. Note that γ 1 -adaptin in these cells is not only present on the structures containing rabaptin-5α (arrows), but also in the TGN area (arrow heads). Saponin treatment of µ 1 A–/– cells removed all γ 1 -adaptin labelling (not shown). Cells were fixed, and labelled with a rabbit antibody against rabaptin-5α (red) and mouse γ 1 -adaptin antibody (Transduction labs) (green). Bar, 10 µm.
    Pcdna3 1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 40640 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcdna3 1/product/Thermo Fisher
    Average 99 stars, based on 40640 article reviews
    Price from $9.99 to $1999.99
    pcdna3 1 - by Bioz Stars, 2020-02
    99/100 stars
      Buy from Supplier

    88
    Thermo Fisher pcdna3 1 a
    Fig. 4. γ 1 –σ 1 AP-1 subcomplex is retained on rabaptin-5α beads. ( A ) GST or GST–rabaptin-5α(301–592) were incubated with glutathione–Sepharose and a HeLa cell extract. The western blot of bound (B) and non-bound (NB) fractions was probed with antibodies against γ 1 , β 1/2 , σ 1 , µ 1 and clathrin. ( B ) IF of clathrin and rabaptin-5α in cells transfected with rabaptin-5α or co-transfected with rabaptin-5α and γ 1 -adaptin. Cells were permeabilized, fixed and labelled with a rabbit antibody against rabaptin-5α (green) and X-22 against clathrin (red). ( C ) HeLa cells were labelled with [ 35 S]methionine and the lysate was incubated with GST–rabaptin-5α(301–592). Bound material was eluted with glutathione. Eluate and cell lysate were incubated with antibodies against γ 1 (100/3), β 1/2 (100/1), µ 1 and σ 1 , and protein A beads. Immunoprecipitates were resolved by SDS–PAGE and analysed by phosphorimaging. To detect σ 1 in the GSH eluate, gels were exposed 10 times longer than for γ 1 -adaptin (σ1 contains four times less methionine residues than γ 1 -adaptin). ( D ) Rabaptin-5α and γ 1 -adaptin co-localize on endosomes in the absence of intact AP-1. µ 1 A–/– and µ 1 A+/+ fibroblasts were transfected with <t>rabaptin-5α-pcDNA3.1His.</t> For γ 1 -adaptin labelling in µ 1 A+/+ fibroblasts, cells were extracted with saponin before fixation. Note that γ 1 -adaptin in these cells is not only present on the structures containing rabaptin-5α (arrows), but also in the TGN area (arrow heads). Saponin treatment of µ 1 A–/– cells removed all γ 1 -adaptin labelling (not shown). Cells were fixed, and labelled with a rabbit antibody against rabaptin-5α (red) and mouse γ 1 -adaptin antibody (Transduction labs) (green). Bar, 10 µm.
    Pcdna3 1 A, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 41 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcdna3 1 a/product/Thermo Fisher
    Average 88 stars, based on 41 article reviews
    Price from $9.99 to $1999.99
    pcdna3 1 a - by Bioz Stars, 2020-02
    88/100 stars
      Buy from Supplier

    77
    Thermo Fisher pc3 1 topo
    Fig. 4. γ 1 –σ 1 AP-1 subcomplex is retained on rabaptin-5α beads. ( A ) GST or GST–rabaptin-5α(301–592) were incubated with glutathione–Sepharose and a HeLa cell extract. The western blot of bound (B) and non-bound (NB) fractions was probed with antibodies against γ 1 , β 1/2 , σ 1 , µ 1 and clathrin. ( B ) IF of clathrin and rabaptin-5α in cells transfected with rabaptin-5α or co-transfected with rabaptin-5α and γ 1 -adaptin. Cells were permeabilized, fixed and labelled with a rabbit antibody against rabaptin-5α (green) and X-22 against clathrin (red). ( C ) HeLa cells were labelled with [ 35 S]methionine and the lysate was incubated with GST–rabaptin-5α(301–592). Bound material was eluted with glutathione. Eluate and cell lysate were incubated with antibodies against γ 1 (100/3), β 1/2 (100/1), µ 1 and σ 1 , and protein A beads. Immunoprecipitates were resolved by SDS–PAGE and analysed by phosphorimaging. To detect σ 1 in the GSH eluate, gels were exposed 10 times longer than for γ 1 -adaptin (σ1 contains four times less methionine residues than γ 1 -adaptin). ( D ) Rabaptin-5α and γ 1 -adaptin co-localize on endosomes in the absence of intact AP-1. µ 1 A–/– and µ 1 A+/+ fibroblasts were transfected with <t>rabaptin-5α-pcDNA3.1His.</t> For γ 1 -adaptin labelling in µ 1 A+/+ fibroblasts, cells were extracted with saponin before fixation. Note that γ 1 -adaptin in these cells is not only present on the structures containing rabaptin-5α (arrows), but also in the TGN area (arrow heads). Saponin treatment of µ 1 A–/– cells removed all γ 1 -adaptin labelling (not shown). Cells were fixed, and labelled with a rabbit antibody against rabaptin-5α (red) and mouse γ 1 -adaptin antibody (Transduction labs) (green). Bar, 10 µm.
    Pc3 1 Topo, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 77/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pc3 1 topo/product/Thermo Fisher
    Average 77 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    pc3 1 topo - by Bioz Stars, 2020-02
    77/100 stars
      Buy from Supplier

    79
    Thermo Fisher pc3 1 vector
    Overexpression of Rab1 protein antagonizes the inhibitory effect of radicicol on VSV-G ts transport. (A) CHO-K1 cells were transfected with <t>pcDNA3.1</t> plasmids containing Rab1B, Rab3A, Sar1A, or empty vector 24 h before infection with VSV ts as described in Materials and Methods . Infected cells were then pulse-labeled in the absence or presence of radicicol (Rad). Representative autoradiograph is shown. (B) Quantitation based on two independent experiments with triplicate samples. Reported as percentage of increase in endo H-resistant VSV-G ts normalized to mock control to illustrate fold-difference in recovery. (C) Immunoblot analysis of transfected cell lysates demonstrating expression of each of the GTPases relative to endogenous levels. Transient expression did not significantly affect the endogenous level of Hsp90, GDI or alter the total amount of VSV-G ts synthesized.
    Pc3 1 Vector, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 79/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pc3 1 vector/product/Thermo Fisher
    Average 79 stars, based on 11 article reviews
    Price from $9.99 to $1999.99
    pc3 1 vector - by Bioz Stars, 2020-02
    79/100 stars
      Buy from Supplier

    85
    Thermo Fisher pcdna3 1 3
    Overexpression of Rab1 protein antagonizes the inhibitory effect of radicicol on VSV-G ts transport. (A) CHO-K1 cells were transfected with <t>pcDNA3.1</t> plasmids containing Rab1B, Rab3A, Sar1A, or empty vector 24 h before infection with VSV ts as described in Materials and Methods . Infected cells were then pulse-labeled in the absence or presence of radicicol (Rad). Representative autoradiograph is shown. (B) Quantitation based on two independent experiments with triplicate samples. Reported as percentage of increase in endo H-resistant VSV-G ts normalized to mock control to illustrate fold-difference in recovery. (C) Immunoblot analysis of transfected cell lysates demonstrating expression of each of the GTPases relative to endogenous levels. Transient expression did not significantly affect the endogenous level of Hsp90, GDI or alter the total amount of VSV-G ts synthesized.
    Pcdna3 1 3, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcdna3 1 3/product/Thermo Fisher
    Average 85 stars, based on 21 article reviews
    Price from $9.99 to $1999.99
    pcdna3 1 3 - by Bioz Stars, 2020-02
    85/100 stars
      Buy from Supplier

    77
    Thermo Fisher pcdna3 1 verctor
    Overexpression of Rab1 protein antagonizes the inhibitory effect of radicicol on VSV-G ts transport. (A) CHO-K1 cells were transfected with <t>pcDNA3.1</t> plasmids containing Rab1B, Rab3A, Sar1A, or empty vector 24 h before infection with VSV ts as described in Materials and Methods . Infected cells were then pulse-labeled in the absence or presence of radicicol (Rad). Representative autoradiograph is shown. (B) Quantitation based on two independent experiments with triplicate samples. Reported as percentage of increase in endo H-resistant VSV-G ts normalized to mock control to illustrate fold-difference in recovery. (C) Immunoblot analysis of transfected cell lysates demonstrating expression of each of the GTPases relative to endogenous levels. Transient expression did not significantly affect the endogenous level of Hsp90, GDI or alter the total amount of VSV-G ts synthesized.
    Pcdna3 1 Verctor, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 77/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcdna3 1 verctor/product/Thermo Fisher
    Average 77 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    pcdna3 1 verctor - by Bioz Stars, 2020-02
    77/100 stars
      Buy from Supplier

    99
    Thermo Fisher pcdna3 1 zeo
    Analysis of the particle association of the Env-Bet fusion protein. Following transfection with different proviral expression constructs, 293T cells were metabolically labeled, and the appearance of HFV Env proteins in the supernatant and associated with viral particles was analyzed. (A) RIPA of viral proteins secreted into the supernatant by using an anti-HFV chimpanzee serum. On the left is a longer exposure of lanes 1 and 2. (B) Particle-associated proteins after pelleting through 20% sucrose. Lanes: 1, pcHSRV2 wt; 2, pcHSRV2 EM4; 3, pcHSRV2 EM6; 4, pcHSRV2 EM7; 5, pcHSRV2 EM8; 6, pcHSRV2 EM9; 7, mock, <t>pCDNA3.1+zeo.</t>
    Pcdna3 1 Zeo, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 788 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcdna3 1 zeo/product/Thermo Fisher
    Average 99 stars, based on 788 article reviews
    Price from $9.99 to $1999.99
    pcdna3 1 zeo - by Bioz Stars, 2020-02
    99/100 stars
      Buy from Supplier

    99
    Thermo Fisher pcdna3 1 hygro
    Identification of splice variants of canine mda- 7. Canine mda- 7 splice variants were amplified by nested PCR and cloned into the <t>pCDNA3.1+/Hygro</t> plasmid vector. Recombinant clones were digested with Hind III and Xho I restriction enzymes to release the
    Pcdna3 1 Hygro, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 708 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcdna3 1 hygro/product/Thermo Fisher
    Average 99 stars, based on 708 article reviews
    Price from $9.99 to $1999.99
    pcdna3 1 hygro - by Bioz Stars, 2020-02
    99/100 stars
      Buy from Supplier

    94
    Thermo Fisher pcdna3 1 v5
    Effects of NSPc1 on HeLa cell cycle progression, cell proliferation and cell colony formation. ( a ) Establishment of the NSPc1 stably over-expressed and NSPc1 knocked-down HeLa cell pools. Up: changes in mRNA level of HeLa stably transfected cells pool confirmed by semi-quantitative RT–PCR. PCR products generated by primer sets for NSPc1 were normalized by glyceraldehyde-3-phosphate dehydrogenase (GAPDH); Down: changes in protein expression confirmed by Western blots (normalized by β-actin protein level). ( b and c ) Flow cytometric analysis of HeLa stable cell pools. ( d and e ) MTT assay of HeLa stable cell pools. ( f and g ) Colony formation assay. Culture dishes fixed and stained with 0.1% crystal violet at the end of the experiment. The numbers of total colonies and colonies greater than 2 mm in diameter were counted and the means and SDs of three independent experiments were shown. Abbreviations: V5, <t>pcDNA3.1-V5</t> cell pool; NSPc1, pcDNA3.1-V5+NSPc1 cell pool; IMG800, IMG800 cell pool; RNAi1, NSPc1-RNAi1 cell pool; RNAi2, NSPc1-RNAi2 cell pool. Note: a representative experiment out of three performed is shown in (b–f).
    Pcdna3 1 V5, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 302 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcdna3 1 v5/product/Thermo Fisher
    Average 94 stars, based on 302 article reviews
    Price from $9.99 to $1999.99
    pcdna3 1 v5 - by Bioz Stars, 2020-02
    94/100 stars
      Buy from Supplier

    77
    Thermo Fisher i pcdna3 1
    Effects of NSPc1 on HeLa cell cycle progression, cell proliferation and cell colony formation. ( a ) Establishment of the NSPc1 stably over-expressed and NSPc1 knocked-down HeLa cell pools. Up: changes in mRNA level of HeLa stably transfected cells pool confirmed by semi-quantitative RT–PCR. PCR products generated by primer sets for NSPc1 were normalized by glyceraldehyde-3-phosphate dehydrogenase (GAPDH); Down: changes in protein expression confirmed by Western blots (normalized by β-actin protein level). ( b and c ) Flow cytometric analysis of HeLa stable cell pools. ( d and e ) MTT assay of HeLa stable cell pools. ( f and g ) Colony formation assay. Culture dishes fixed and stained with 0.1% crystal violet at the end of the experiment. The numbers of total colonies and colonies greater than 2 mm in diameter were counted and the means and SDs of three independent experiments were shown. Abbreviations: V5, <t>pcDNA3.1-V5</t> cell pool; NSPc1, pcDNA3.1-V5+NSPc1 cell pool; IMG800, IMG800 cell pool; RNAi1, NSPc1-RNAi1 cell pool; RNAi2, NSPc1-RNAi2 cell pool. Note: a representative experiment out of three performed is shown in (b–f).
    I Pcdna3 1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 77/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/i pcdna3 1/product/Thermo Fisher
    Average 77 stars, based on 5 article reviews
    Price from $9.99 to $1999.99
    i pcdna3 1 - by Bioz Stars, 2020-02
    77/100 stars
      Buy from Supplier

    99
    Thermo Fisher empty pcdna3 1
    FUS promotes miR-200c-mediated degradation of ZEB1 (A) Bar graphs show the amount of immunoprecipitated miR-200c (n=5). HEK293 cells are transfected with myc-FUS, myc-R495X, myc-SOD1, or control <t>pcDNA3.1,</t> along with pCMV-miR-200c, prior to the immunoprecipitation by anti-myc antibodies. (B) Bar graph represents enrichment of mature miR-200c in RIP experiments using a FUS antibody in human WT HAP1 cells versus FUS KO HAP1 cells (n=3), indicating the endogenous interaction between FUS and miR-200c. (C) Bar graphs show the amount of immunoprecipitated mRNA of ZEB1 (left) and FZD6 (right). HEK293 cells are transfected with pcDNA3.1, myc-FUS or myc-R495X, along with pCMV-miR-200c, prior to immunoprecipitation with anti-myc antibodies. (D) Bar graphs show the amount of immunoprecipitated mRNA of ZEB1 in HEK293 cells transfected with FUS-V5 or LacZ-V5 together with a specific miR-200c antagomir or a control antagomir, prior to immunoprecipitation with anti-V5 antibodies. (E) (top) A model of the experimental design to test the role of FUS or miR-200c binding to ZEB1 3′ UTR in its silencing. Bar graphs compare the normalized luciferase activity of the pmirGLO reporters with WT or mutated ZEB1 3′ UTRs in the presence of recombinant pCMV-miR-505 or pCMV-miR-200c, plotted as a percentage relative to the control (n=3). Normalization was performed by dividing the firefly activity by the Renilla .
    Empty Pcdna3 1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 368 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/empty pcdna3 1/product/Thermo Fisher
    Average 99 stars, based on 368 article reviews
    Price from $9.99 to $1999.99
    empty pcdna3 1 - by Bioz Stars, 2020-02
    99/100 stars
      Buy from Supplier

    78
    Thermo Fisher comp pcdna3 1
    FUS promotes miR-200c-mediated degradation of ZEB1 (A) Bar graphs show the amount of immunoprecipitated miR-200c (n=5). HEK293 cells are transfected with myc-FUS, myc-R495X, myc-SOD1, or control <t>pcDNA3.1,</t> along with pCMV-miR-200c, prior to the immunoprecipitation by anti-myc antibodies. (B) Bar graph represents enrichment of mature miR-200c in RIP experiments using a FUS antibody in human WT HAP1 cells versus FUS KO HAP1 cells (n=3), indicating the endogenous interaction between FUS and miR-200c. (C) Bar graphs show the amount of immunoprecipitated mRNA of ZEB1 (left) and FZD6 (right). HEK293 cells are transfected with pcDNA3.1, myc-FUS or myc-R495X, along with pCMV-miR-200c, prior to immunoprecipitation with anti-myc antibodies. (D) Bar graphs show the amount of immunoprecipitated mRNA of ZEB1 in HEK293 cells transfected with FUS-V5 or LacZ-V5 together with a specific miR-200c antagomir or a control antagomir, prior to immunoprecipitation with anti-V5 antibodies. (E) (top) A model of the experimental design to test the role of FUS or miR-200c binding to ZEB1 3′ UTR in its silencing. Bar graphs compare the normalized luciferase activity of the pmirGLO reporters with WT or mutated ZEB1 3′ UTRs in the presence of recombinant pCMV-miR-505 or pCMV-miR-200c, plotted as a percentage relative to the control (n=3). Normalization was performed by dividing the firefly activity by the Renilla .
    Comp Pcdna3 1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 78/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/comp pcdna3 1/product/Thermo Fisher
    Average 78 stars, based on 9 article reviews
    Price from $9.99 to $1999.99
    comp pcdna3 1 - by Bioz Stars, 2020-02
    78/100 stars
      Buy from Supplier

    79
    Thermo Fisher eukaryotic pcdna3 1
    FUS promotes miR-200c-mediated degradation of ZEB1 (A) Bar graphs show the amount of immunoprecipitated miR-200c (n=5). HEK293 cells are transfected with myc-FUS, myc-R495X, myc-SOD1, or control <t>pcDNA3.1,</t> along with pCMV-miR-200c, prior to the immunoprecipitation by anti-myc antibodies. (B) Bar graph represents enrichment of mature miR-200c in RIP experiments using a FUS antibody in human WT HAP1 cells versus FUS KO HAP1 cells (n=3), indicating the endogenous interaction between FUS and miR-200c. (C) Bar graphs show the amount of immunoprecipitated mRNA of ZEB1 (left) and FZD6 (right). HEK293 cells are transfected with pcDNA3.1, myc-FUS or myc-R495X, along with pCMV-miR-200c, prior to immunoprecipitation with anti-myc antibodies. (D) Bar graphs show the amount of immunoprecipitated mRNA of ZEB1 in HEK293 cells transfected with FUS-V5 or LacZ-V5 together with a specific miR-200c antagomir or a control antagomir, prior to immunoprecipitation with anti-V5 antibodies. (E) (top) A model of the experimental design to test the role of FUS or miR-200c binding to ZEB1 3′ UTR in its silencing. Bar graphs compare the normalized luciferase activity of the pmirGLO reporters with WT or mutated ZEB1 3′ UTRs in the presence of recombinant pCMV-miR-505 or pCMV-miR-200c, plotted as a percentage relative to the control (n=3). Normalization was performed by dividing the firefly activity by the Renilla .
    Eukaryotic Pcdna3 1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 79/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/eukaryotic pcdna3 1/product/Thermo Fisher
    Average 79 stars, based on 14 article reviews
    Price from $9.99 to $1999.99
    eukaryotic pcdna3 1 - by Bioz Stars, 2020-02
    79/100 stars
      Buy from Supplier

    79
    Thermo Fisher pc3 1 mychisa vector
    FUS promotes miR-200c-mediated degradation of ZEB1 (A) Bar graphs show the amount of immunoprecipitated miR-200c (n=5). HEK293 cells are transfected with myc-FUS, myc-R495X, myc-SOD1, or control <t>pcDNA3.1,</t> along with pCMV-miR-200c, prior to the immunoprecipitation by anti-myc antibodies. (B) Bar graph represents enrichment of mature miR-200c in RIP experiments using a FUS antibody in human WT HAP1 cells versus FUS KO HAP1 cells (n=3), indicating the endogenous interaction between FUS and miR-200c. (C) Bar graphs show the amount of immunoprecipitated mRNA of ZEB1 (left) and FZD6 (right). HEK293 cells are transfected with pcDNA3.1, myc-FUS or myc-R495X, along with pCMV-miR-200c, prior to immunoprecipitation with anti-myc antibodies. (D) Bar graphs show the amount of immunoprecipitated mRNA of ZEB1 in HEK293 cells transfected with FUS-V5 or LacZ-V5 together with a specific miR-200c antagomir or a control antagomir, prior to immunoprecipitation with anti-V5 antibodies. (E) (top) A model of the experimental design to test the role of FUS or miR-200c binding to ZEB1 3′ UTR in its silencing. Bar graphs compare the normalized luciferase activity of the pmirGLO reporters with WT or mutated ZEB1 3′ UTRs in the presence of recombinant pCMV-miR-505 or pCMV-miR-200c, plotted as a percentage relative to the control (n=3). Normalization was performed by dividing the firefly activity by the Renilla .
    Pc3 1 Mychisa Vector, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 79/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pc3 1 mychisa vector/product/Thermo Fisher
    Average 79 stars, based on 5 article reviews
    Price from $9.99 to $1999.99
    pc3 1 mychisa vector - by Bioz Stars, 2020-02
    79/100 stars
      Buy from Supplier

    77
    Thermo Fisher plasmid pc3 1 zeo
    FUS promotes miR-200c-mediated degradation of ZEB1 (A) Bar graphs show the amount of immunoprecipitated miR-200c (n=5). HEK293 cells are transfected with myc-FUS, myc-R495X, myc-SOD1, or control <t>pcDNA3.1,</t> along with pCMV-miR-200c, prior to the immunoprecipitation by anti-myc antibodies. (B) Bar graph represents enrichment of mature miR-200c in RIP experiments using a FUS antibody in human WT HAP1 cells versus FUS KO HAP1 cells (n=3), indicating the endogenous interaction between FUS and miR-200c. (C) Bar graphs show the amount of immunoprecipitated mRNA of ZEB1 (left) and FZD6 (right). HEK293 cells are transfected with pcDNA3.1, myc-FUS or myc-R495X, along with pCMV-miR-200c, prior to immunoprecipitation with anti-myc antibodies. (D) Bar graphs show the amount of immunoprecipitated mRNA of ZEB1 in HEK293 cells transfected with FUS-V5 or LacZ-V5 together with a specific miR-200c antagomir or a control antagomir, prior to immunoprecipitation with anti-V5 antibodies. (E) (top) A model of the experimental design to test the role of FUS or miR-200c binding to ZEB1 3′ UTR in its silencing. Bar graphs compare the normalized luciferase activity of the pmirGLO reporters with WT or mutated ZEB1 3′ UTRs in the presence of recombinant pCMV-miR-505 or pCMV-miR-200c, plotted as a percentage relative to the control (n=3). Normalization was performed by dividing the firefly activity by the Renilla .
    Plasmid Pc3 1 Zeo, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 77/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/plasmid pc3 1 zeo/product/Thermo Fisher
    Average 77 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    plasmid pc3 1 zeo - by Bioz Stars, 2020-02
    77/100 stars
      Buy from Supplier

    94
    Thermo Fisher pcdna3 1 n ha
    FUS promotes miR-200c-mediated degradation of ZEB1 (A) Bar graphs show the amount of immunoprecipitated miR-200c (n=5). HEK293 cells are transfected with myc-FUS, myc-R495X, myc-SOD1, or control <t>pcDNA3.1,</t> along with pCMV-miR-200c, prior to the immunoprecipitation by anti-myc antibodies. (B) Bar graph represents enrichment of mature miR-200c in RIP experiments using a FUS antibody in human WT HAP1 cells versus FUS KO HAP1 cells (n=3), indicating the endogenous interaction between FUS and miR-200c. (C) Bar graphs show the amount of immunoprecipitated mRNA of ZEB1 (left) and FZD6 (right). HEK293 cells are transfected with pcDNA3.1, myc-FUS or myc-R495X, along with pCMV-miR-200c, prior to immunoprecipitation with anti-myc antibodies. (D) Bar graphs show the amount of immunoprecipitated mRNA of ZEB1 in HEK293 cells transfected with FUS-V5 or LacZ-V5 together with a specific miR-200c antagomir or a control antagomir, prior to immunoprecipitation with anti-V5 antibodies. (E) (top) A model of the experimental design to test the role of FUS or miR-200c binding to ZEB1 3′ UTR in its silencing. Bar graphs compare the normalized luciferase activity of the pmirGLO reporters with WT or mutated ZEB1 3′ UTRs in the presence of recombinant pCMV-miR-505 or pCMV-miR-200c, plotted as a percentage relative to the control (n=3). Normalization was performed by dividing the firefly activity by the Renilla .
    Pcdna3 1 N Ha, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcdna3 1 n ha/product/Thermo Fisher
    Average 94 stars, based on 21 article reviews
    Price from $9.99 to $1999.99
    pcdna3 1 n ha - by Bioz Stars, 2020-02
    94/100 stars
      Buy from Supplier

    99
    Thermo Fisher plasmid pcdna3 1
    Constitutively expressed MEG3 suppresses xenograft tumor growth in nude mice. PDFS cells were transfected with <t>pcDNA3.1-puro</t> vector, pCMV-MEG3-puro or pCMV-MEG3del5-puro constructs. Puromycin resistant clones were isolated. MEG3 (A) or MEG3del5 (B) expression
    Plasmid Pcdna3 1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 888 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/plasmid pcdna3 1/product/Thermo Fisher
    Average 99 stars, based on 888 article reviews
    Price from $9.99 to $1999.99
    plasmid pcdna3 1 - by Bioz Stars, 2020-02
    99/100 stars
      Buy from Supplier

    77
    Thermo Fisher egfp tagged pcdna3 1
    Constitutively expressed MEG3 suppresses xenograft tumor growth in nude mice. PDFS cells were transfected with <t>pcDNA3.1-puro</t> vector, pCMV-MEG3-puro or pCMV-MEG3del5-puro constructs. Puromycin resistant clones were isolated. MEG3 (A) or MEG3del5 (B) expression
    Egfp Tagged Pcdna3 1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 77/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/egfp tagged pcdna3 1/product/Thermo Fisher
    Average 77 stars, based on 5 article reviews
    Price from $9.99 to $1999.99
    egfp tagged pcdna3 1 - by Bioz Stars, 2020-02
    77/100 stars
      Buy from Supplier

    77
    Thermo Fisher ecori cut pcdna3 1
    Constitutively expressed MEG3 suppresses xenograft tumor growth in nude mice. PDFS cells were transfected with <t>pcDNA3.1-puro</t> vector, pCMV-MEG3-puro or pCMV-MEG3del5-puro constructs. Puromycin resistant clones were isolated. MEG3 (A) or MEG3del5 (B) expression
    Ecori Cut Pcdna3 1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 77/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ecori cut pcdna3 1/product/Thermo Fisher
    Average 77 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    ecori cut pcdna3 1 - by Bioz Stars, 2020-02
    77/100 stars
      Buy from Supplier

    90
    Thermo Fisher pcdna3 1 hygromycin
    Constitutively expressed MEG3 suppresses xenograft tumor growth in nude mice. PDFS cells were transfected with <t>pcDNA3.1-puro</t> vector, pCMV-MEG3-puro or pCMV-MEG3del5-puro constructs. Puromycin resistant clones were isolated. MEG3 (A) or MEG3del5 (B) expression
    Pcdna3 1 Hygromycin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 42 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcdna3 1 hygromycin/product/Thermo Fisher
    Average 90 stars, based on 42 article reviews
    Price from $9.99 to $1999.99
    pcdna3 1 hygromycin - by Bioz Stars, 2020-02
    90/100 stars
      Buy from Supplier

    89
    Thermo Fisher flag tagged pcdna3 1
    Constitutively expressed MEG3 suppresses xenograft tumor growth in nude mice. PDFS cells were transfected with <t>pcDNA3.1-puro</t> vector, pCMV-MEG3-puro or pCMV-MEG3del5-puro constructs. Puromycin resistant clones were isolated. MEG3 (A) or MEG3del5 (B) expression
    Flag Tagged Pcdna3 1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 89/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/flag tagged pcdna3 1/product/Thermo Fisher
    Average 89 stars, based on 29 article reviews
    Price from $9.99 to $1999.99
    flag tagged pcdna3 1 - by Bioz Stars, 2020-02
    89/100 stars
      Buy from Supplier

    79
    Thermo Fisher pcdna3 1 topota system
    Constitutively expressed MEG3 suppresses xenograft tumor growth in nude mice. PDFS cells were transfected with <t>pcDNA3.1-puro</t> vector, pCMV-MEG3-puro or pCMV-MEG3del5-puro constructs. Puromycin resistant clones were isolated. MEG3 (A) or MEG3del5 (B) expression
    Pcdna3 1 Topota System, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 79/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcdna3 1 topota system/product/Thermo Fisher
    Average 79 stars, based on 11 article reviews
    Price from $9.99 to $1999.99
    pcdna3 1 topota system - by Bioz Stars, 2020-02
    79/100 stars
      Buy from Supplier

    78
    Thermo Fisher pcdna3 1 nflag dest
    Constitutively expressed MEG3 suppresses xenograft tumor growth in nude mice. PDFS cells were transfected with <t>pcDNA3.1-puro</t> vector, pCMV-MEG3-puro or pCMV-MEG3del5-puro constructs. Puromycin resistant clones were isolated. MEG3 (A) or MEG3del5 (B) expression
    Pcdna3 1 Nflag Dest, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 78/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcdna3 1 nflag dest/product/Thermo Fisher
    Average 78 stars, based on 6 article reviews
    Price from $9.99 to $1999.99
    pcdna3 1 nflag dest - by Bioz Stars, 2020-02
    78/100 stars
      Buy from Supplier

    79
    Thermo Fisher noti digested pcdna3 1
    Constitutively expressed MEG3 suppresses xenograft tumor growth in nude mice. PDFS cells were transfected with <t>pcDNA3.1-puro</t> vector, pCMV-MEG3-puro or pCMV-MEG3del5-puro constructs. Puromycin resistant clones were isolated. MEG3 (A) or MEG3del5 (B) expression
    Noti Digested Pcdna3 1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 79/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/noti digested pcdna3 1/product/Thermo Fisher
    Average 79 stars, based on 12 article reviews
    Price from $9.99 to $1999.99
    noti digested pcdna3 1 - by Bioz Stars, 2020-02
    79/100 stars
      Buy from Supplier

    76
    Thermo Fisher nhei ecorv pcdna3 1
    Constitutively expressed MEG3 suppresses xenograft tumor growth in nude mice. PDFS cells were transfected with <t>pcDNA3.1-puro</t> vector, pCMV-MEG3-puro or pCMV-MEG3del5-puro constructs. Puromycin resistant clones were isolated. MEG3 (A) or MEG3del5 (B) expression
    Nhei Ecorv Pcdna3 1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 76/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/nhei ecorv pcdna3 1/product/Thermo Fisher
    Average 76 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    nhei ecorv pcdna3 1 - by Bioz Stars, 2020-02
    76/100 stars
      Buy from Supplier

    80
    Thermo Fisher full length pcdna3 1
    Effect of baicalein on the ERK pathway. (A) The protein levels of MEK1, p-MEK1, ERK1/2 and p-ERK1/2. (B) Phosphorylation densities of MEK1 and ERK1/2 were digitally scanned. (C) Transwell assays were performed to evaluate the anti-metastatic effects of baicalein on two groups of cells transfected with the indicated plasmids (transfected with the empty vector <t>pcDNA3.1</t> (+) or with pcDNA3.1 (+)-MEK1, ‘pcDNA3.1(+)’ means the group transfected with an empty vector pcDNA3.1(+), ‘pcDNA3.1(+)-MEK1’ means the group transfected with a pcDNA3.1(+)-MEK1). (D) The percent invasion rate was expressed as a percentage of the control (the control group of cells transfected with an empty vector pcDNA3.1 (+)). (E) The inhibition rates of baicalein on two groups of cells. Values represent the means ± SD of three independent experiments performed in triplicate. * p
    Full Length Pcdna3 1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 80/100, based on 33 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/full length pcdna3 1/product/Thermo Fisher
    Average 80 stars, based on 33 article reviews
    Price from $9.99 to $1999.99
    full length pcdna3 1 - by Bioz Stars, 2020-02
    80/100 stars
      Buy from Supplier

    88
    Thermo Fisher pcdna3 1 v5 hisa
    Evaluation of in vitro DNA binding of Hey1 to myogenic promoter elements. A , TNT lysates programmed with either <t>pcDNA3.1-V5/HisA</t> empty vector or pcDNA3.1-TOPO-Hey1-V5 were incubated with a 22-nucleotide 32 P-labeled probe containing the Hey1 consensus target E-box (CACGTG). Anti-V5 antibodies and cold competitor probes (50× excess relative to labeled probe) were added as indicated before resolution of complexes by non-denaturing SDS-PAGE.; Δ HCE , mutant Hey1-consensus E-box; E1 , E2 , E-boxes within the myogenin proximal promoter; N1 , N-box ∼400 bp upstream of myogenin start site; 2C , high affinity MyoD E-box within the Mef2C proximal promoter. B , 10T1/2 cells were transfected with 25 ng of pRL-tk-Renilla, 25 ng of G133-luciferase or G133-mutE1-luciferase, 25 ng of pEMSV-MyoD, and 25–100 ng of pcDNA3.1-Hey1-V5. Firefly luciferase values were normalized to Renilla luciferase and plotted as the averages of three replicate samples ±S.D.
    Pcdna3 1 V5 Hisa, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 60 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcdna3 1 v5 hisa/product/Thermo Fisher
    Average 88 stars, based on 60 article reviews
    Price from $9.99 to $1999.99
    pcdna3 1 v5 hisa - by Bioz Stars, 2020-02
    88/100 stars
      Buy from Supplier

    80
    Thermo Fisher pcdna3 1 nv5 dest
    Evaluation of in vitro DNA binding of Hey1 to myogenic promoter elements. A , TNT lysates programmed with either <t>pcDNA3.1-V5/HisA</t> empty vector or pcDNA3.1-TOPO-Hey1-V5 were incubated with a 22-nucleotide 32 P-labeled probe containing the Hey1 consensus target E-box (CACGTG). Anti-V5 antibodies and cold competitor probes (50× excess relative to labeled probe) were added as indicated before resolution of complexes by non-denaturing SDS-PAGE.; Δ HCE , mutant Hey1-consensus E-box; E1 , E2 , E-boxes within the myogenin proximal promoter; N1 , N-box ∼400 bp upstream of myogenin start site; 2C , high affinity MyoD E-box within the Mef2C proximal promoter. B , 10T1/2 cells were transfected with 25 ng of pRL-tk-Renilla, 25 ng of G133-luciferase or G133-mutE1-luciferase, 25 ng of pEMSV-MyoD, and 25–100 ng of pcDNA3.1-Hey1-V5. Firefly luciferase values were normalized to Renilla luciferase and plotted as the averages of three replicate samples ±S.D.
    Pcdna3 1 Nv5 Dest, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 80/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcdna3 1 nv5 dest/product/Thermo Fisher
    Average 80 stars, based on 19 article reviews
    Price from $9.99 to $1999.99
    pcdna3 1 nv5 dest - by Bioz Stars, 2020-02
    80/100 stars
      Buy from Supplier

    78
    Thermo Fisher xhoi digested pcdna3 1
    Evaluation of in vitro DNA binding of Hey1 to myogenic promoter elements. A , TNT lysates programmed with either <t>pcDNA3.1-V5/HisA</t> empty vector or pcDNA3.1-TOPO-Hey1-V5 were incubated with a 22-nucleotide 32 P-labeled probe containing the Hey1 consensus target E-box (CACGTG). Anti-V5 antibodies and cold competitor probes (50× excess relative to labeled probe) were added as indicated before resolution of complexes by non-denaturing SDS-PAGE.; Δ HCE , mutant Hey1-consensus E-box; E1 , E2 , E-boxes within the myogenin proximal promoter; N1 , N-box ∼400 bp upstream of myogenin start site; 2C , high affinity MyoD E-box within the Mef2C proximal promoter. B , 10T1/2 cells were transfected with 25 ng of pRL-tk-Renilla, 25 ng of G133-luciferase or G133-mutE1-luciferase, 25 ng of pEMSV-MyoD, and 25–100 ng of pcDNA3.1-Hey1-V5. Firefly luciferase values were normalized to Renilla luciferase and plotted as the averages of three replicate samples ±S.D.
    Xhoi Digested Pcdna3 1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 78/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/xhoi digested pcdna3 1/product/Thermo Fisher
    Average 78 stars, based on 7 article reviews
    Price from $9.99 to $1999.99
    xhoi digested pcdna3 1 - by Bioz Stars, 2020-02
    78/100 stars
      Buy from Supplier

    78
    Thermo Fisher control pcdna3 1 mock
    COSMC modulates protein degradation of VEGFR2. (A) COSMC overexpression delays degradation of VEGFR2. HUVECs were treated with cycloheximide (10 µg/ml) to block protein synthesis and 20 ng/ml of VEGF to trigger internalization and degradation of VEGFR2 for indicated time points. Upper panel shows representative Western blots. Lower panel shows signals on Western blots quantified by ImageQuant5.1 for HUVECs transfected with <t>pcDNA3.1</t> control plasmid (dashed line) and UVECs transfected with COSMC/pcDNA3.1 (solid line). (B) COSMC knockdown facilitates degradation of VEGFR2. VEGFR2 degradation in HUVECs transfected with control siRNA (dashed line) or COSMC siRNA (solid line) was shown. Representative data from two independent experiments are presented.
    Control Pcdna3 1 Mock, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 78/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/control pcdna3 1 mock/product/Thermo Fisher
    Average 78 stars, based on 10 article reviews
    Price from $9.99 to $1999.99
    control pcdna3 1 mock - by Bioz Stars, 2020-02
    78/100 stars
      Buy from Supplier

    82
    Thermo Fisher nhei digested pcdna3 1
    COSMC modulates protein degradation of VEGFR2. (A) COSMC overexpression delays degradation of VEGFR2. HUVECs were treated with cycloheximide (10 µg/ml) to block protein synthesis and 20 ng/ml of VEGF to trigger internalization and degradation of VEGFR2 for indicated time points. Upper panel shows representative Western blots. Lower panel shows signals on Western blots quantified by ImageQuant5.1 for HUVECs transfected with <t>pcDNA3.1</t> control plasmid (dashed line) and UVECs transfected with COSMC/pcDNA3.1 (solid line). (B) COSMC knockdown facilitates degradation of VEGFR2. VEGFR2 degradation in HUVECs transfected with control siRNA (dashed line) or COSMC siRNA (solid line) was shown. Representative data from two independent experiments are presented.
    Nhei Digested Pcdna3 1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 82/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/nhei digested pcdna3 1/product/Thermo Fisher
    Average 82 stars, based on 17 article reviews
    Price from $9.99 to $1999.99
    nhei digested pcdna3 1 - by Bioz Stars, 2020-02
    82/100 stars
      Buy from Supplier

    Image Search Results


    Fig. 4. γ 1 –σ 1 AP-1 subcomplex is retained on rabaptin-5α beads. ( A ) GST or GST–rabaptin-5α(301–592) were incubated with glutathione–Sepharose and a HeLa cell extract. The western blot of bound (B) and non-bound (NB) fractions was probed with antibodies against γ 1 , β 1/2 , σ 1 , µ 1 and clathrin. ( B ) IF of clathrin and rabaptin-5α in cells transfected with rabaptin-5α or co-transfected with rabaptin-5α and γ 1 -adaptin. Cells were permeabilized, fixed and labelled with a rabbit antibody against rabaptin-5α (green) and X-22 against clathrin (red). ( C ) HeLa cells were labelled with [ 35 S]methionine and the lysate was incubated with GST–rabaptin-5α(301–592). Bound material was eluted with glutathione. Eluate and cell lysate were incubated with antibodies against γ 1 (100/3), β 1/2 (100/1), µ 1 and σ 1 , and protein A beads. Immunoprecipitates were resolved by SDS–PAGE and analysed by phosphorimaging. To detect σ 1 in the GSH eluate, gels were exposed 10 times longer than for γ 1 -adaptin (σ1 contains four times less methionine residues than γ 1 -adaptin). ( D ) Rabaptin-5α and γ 1 -adaptin co-localize on endosomes in the absence of intact AP-1. µ 1 A–/– and µ 1 A+/+ fibroblasts were transfected with rabaptin-5α-pcDNA3.1His. For γ 1 -adaptin labelling in µ 1 A+/+ fibroblasts, cells were extracted with saponin before fixation. Note that γ 1 -adaptin in these cells is not only present on the structures containing rabaptin-5α (arrows), but also in the TGN area (arrow heads). Saponin treatment of µ 1 A–/– cells removed all γ 1 -adaptin labelling (not shown). Cells were fixed, and labelled with a rabbit antibody against rabaptin-5α (red) and mouse γ 1 -adaptin antibody (Transduction labs) (green). Bar, 10 µm.

    Journal: The EMBO Journal

    Article Title: Rabaptin-5?/rabaptin-4 serves as a linker between rab4 and ?1-adaptin in membrane recycling from endosomes

    doi: 10.1093/emboj/cdg257

    Figure Lengend Snippet: Fig. 4. γ 1 –σ 1 AP-1 subcomplex is retained on rabaptin-5α beads. ( A ) GST or GST–rabaptin-5α(301–592) were incubated with glutathione–Sepharose and a HeLa cell extract. The western blot of bound (B) and non-bound (NB) fractions was probed with antibodies against γ 1 , β 1/2 , σ 1 , µ 1 and clathrin. ( B ) IF of clathrin and rabaptin-5α in cells transfected with rabaptin-5α or co-transfected with rabaptin-5α and γ 1 -adaptin. Cells were permeabilized, fixed and labelled with a rabbit antibody against rabaptin-5α (green) and X-22 against clathrin (red). ( C ) HeLa cells were labelled with [ 35 S]methionine and the lysate was incubated with GST–rabaptin-5α(301–592). Bound material was eluted with glutathione. Eluate and cell lysate were incubated with antibodies against γ 1 (100/3), β 1/2 (100/1), µ 1 and σ 1 , and protein A beads. Immunoprecipitates were resolved by SDS–PAGE and analysed by phosphorimaging. To detect σ 1 in the GSH eluate, gels were exposed 10 times longer than for γ 1 -adaptin (σ1 contains four times less methionine residues than γ 1 -adaptin). ( D ) Rabaptin-5α and γ 1 -adaptin co-localize on endosomes in the absence of intact AP-1. µ 1 A–/– and µ 1 A+/+ fibroblasts were transfected with rabaptin-5α-pcDNA3.1His. For γ 1 -adaptin labelling in µ 1 A+/+ fibroblasts, cells were extracted with saponin before fixation. Note that γ 1 -adaptin in these cells is not only present on the structures containing rabaptin-5α (arrows), but also in the TGN area (arrow heads). Saponin treatment of µ 1 A–/– cells removed all γ 1 -adaptin labelling (not shown). Cells were fixed, and labelled with a rabbit antibody against rabaptin-5α (red) and mouse γ 1 -adaptin antibody (Transduction labs) (green). Bar, 10 µm.

    Article Snippet: Rabaptin constructs were cloned in pcDNA3 and pcDNA3.1His (Invitrogen, Leek, The Netherlands).

    Techniques: Incubation, Western Blot, Transfection, SDS Page, Transduction

    Fig. 2. Rabaptin-5α–γ-adaptin complex is localized on endosomes. HeLa cells were transfected with rabaptin-5α-pcDNA3.1His (left panels) or with rabaptin-5α and γ 1 -adaptin-pcDNA3 (right panels). Transfected cells were labelled for rabaptin-5α (green) and γ 1 -adaptin (red). Note the co-localization of rabaptin-5α and γ 1 -adaptin in transfected cells, and the distinct localization of γ 1 -adaptin in non-transfected cells (arrow) ( A and A ′). Cells were incubated with Alexa594-Tf for 60 min at 37°C and subsequently labelled with anti-Xpress antibody and Alexa488-conjugated IgG ( B and B ′). The TGN marker TGN46 does not relocate to enlarged endosomes. Transfected cells were labelled for rabaptin-5α (red) and TGN46 (green) ( C and C ′). Bar, 10 µm.

    Journal: The EMBO Journal

    Article Title: Rabaptin-5?/rabaptin-4 serves as a linker between rab4 and ?1-adaptin in membrane recycling from endosomes

    doi: 10.1093/emboj/cdg257

    Figure Lengend Snippet: Fig. 2. Rabaptin-5α–γ-adaptin complex is localized on endosomes. HeLa cells were transfected with rabaptin-5α-pcDNA3.1His (left panels) or with rabaptin-5α and γ 1 -adaptin-pcDNA3 (right panels). Transfected cells were labelled for rabaptin-5α (green) and γ 1 -adaptin (red). Note the co-localization of rabaptin-5α and γ 1 -adaptin in transfected cells, and the distinct localization of γ 1 -adaptin in non-transfected cells (arrow) ( A and A ′). Cells were incubated with Alexa594-Tf for 60 min at 37°C and subsequently labelled with anti-Xpress antibody and Alexa488-conjugated IgG ( B and B ′). The TGN marker TGN46 does not relocate to enlarged endosomes. Transfected cells were labelled for rabaptin-5α (red) and TGN46 (green) ( C and C ′). Bar, 10 µm.

    Article Snippet: Rabaptin constructs were cloned in pcDNA3 and pcDNA3.1His (Invitrogen, Leek, The Netherlands).

    Techniques: Transfection, Incubation, Marker

    Fig. 6. Transfected rabaptin-5α delays Tf recycling. ( A ) HeLa cells were transfected with rabaptin-5α-pcDNA3.1His or with rabaptin-5α-pcDNA3.1His and γ 1 -adaptin-pcDNA3. The cells were incubated with 15 µg/ml Alexa-Tf at 16°C for 30 min, and subsequently chased at 37°C. Cells were fixed after different periods of time and stained for rabaptin-5α. Quantitation of the fraction of rabaptin-5α-positive endosomes containing Alexa488-Tf at 0, 10 and 50 min of chase. Error bars denote the standard deviation ( n = 10). ( B ) Rab4 and γ 1 -adaptin interaction domains in rabaptin-5α are required to retard Tf recycling. HeLa cells were transfected with rabaptin-5α(1–390), rabaptin-5α(301–592), rabaptin-5α(1–592) and rabaptin-5α. The cells were subjected to the pulse–chase protocol as above, fixed and labelled with anti-Xpress followed by Alexa488-labelled anti-mouse IgG. Note that only the rabaptin-5α truncation containing both rab4- and γ 1 -adaptin-binding sites retarded Tf recycling. Bar, 10 µm.

    Journal: The EMBO Journal

    Article Title: Rabaptin-5?/rabaptin-4 serves as a linker between rab4 and ?1-adaptin in membrane recycling from endosomes

    doi: 10.1093/emboj/cdg257

    Figure Lengend Snippet: Fig. 6. Transfected rabaptin-5α delays Tf recycling. ( A ) HeLa cells were transfected with rabaptin-5α-pcDNA3.1His or with rabaptin-5α-pcDNA3.1His and γ 1 -adaptin-pcDNA3. The cells were incubated with 15 µg/ml Alexa-Tf at 16°C for 30 min, and subsequently chased at 37°C. Cells were fixed after different periods of time and stained for rabaptin-5α. Quantitation of the fraction of rabaptin-5α-positive endosomes containing Alexa488-Tf at 0, 10 and 50 min of chase. Error bars denote the standard deviation ( n = 10). ( B ) Rab4 and γ 1 -adaptin interaction domains in rabaptin-5α are required to retard Tf recycling. HeLa cells were transfected with rabaptin-5α(1–390), rabaptin-5α(301–592), rabaptin-5α(1–592) and rabaptin-5α. The cells were subjected to the pulse–chase protocol as above, fixed and labelled with anti-Xpress followed by Alexa488-labelled anti-mouse IgG. Note that only the rabaptin-5α truncation containing both rab4- and γ 1 -adaptin-binding sites retarded Tf recycling. Bar, 10 µm.

    Article Snippet: Rabaptin constructs were cloned in pcDNA3 and pcDNA3.1His (Invitrogen, Leek, The Netherlands).

    Techniques: Transfection, Incubation, Staining, Quantitation Assay, Standard Deviation, Pulse Chase, Binding Assay

    Fig. 3. Rabaptin-5α and γ-adaptin co-localize to tubulo-vesicular membrane clusters. Ultrathin cryosections of HeLa cells transfected with rabaptin- 5α-pcDNA3.1His (A,C and E) or with rabaptin-5α and γ 1 -adaptin-pcDNA3 (B and D). Double labelling of rabaptin-5α (10 nm gold) and γ 1 -adaptin (15 nm gold). Rabaptin-5α is associated with clusters of vesicular tubular membranes, where it co-localized with endogenous ( A ) and overexpressed γ 1 -adaptin ( B ). The overall density and diameter of rabaptin-5α-positive membranes is reminiscent of recycling tubules. The dense cytosol between the membranes indicates the presence of high concentrations of cytosolic protein. Note that some of the membranes display a coating typical of the presence of clathrin (arrows in A and B). Double labelling of TGN46 (15 nm gold) and rabaptin-5α (10 nm gold) revealed that rabaptin-5α-positive membranes do not overlap with TGN membranes ( C ). Rabaptin-5α- (15 nm gold) positive membranes do not contain internalized BSA–5 nm gold. Arrows point to compartments that contain the endocytic marker 10 min after internalization ( D ). Double labelling of rabaptin-5α (15 nm gold) and clathrin (10 nm gold). Some of the membranes within or associated with rabaptin-5α-positive membranes also stained for clathrin (arrows). Note that the nearby endosomal vacuole (E) has a normal morphology ( E ). G = Golgi complex, L = lysosome. Bar, 200 nm.

    Journal: The EMBO Journal

    Article Title: Rabaptin-5?/rabaptin-4 serves as a linker between rab4 and ?1-adaptin in membrane recycling from endosomes

    doi: 10.1093/emboj/cdg257

    Figure Lengend Snippet: Fig. 3. Rabaptin-5α and γ-adaptin co-localize to tubulo-vesicular membrane clusters. Ultrathin cryosections of HeLa cells transfected with rabaptin- 5α-pcDNA3.1His (A,C and E) or with rabaptin-5α and γ 1 -adaptin-pcDNA3 (B and D). Double labelling of rabaptin-5α (10 nm gold) and γ 1 -adaptin (15 nm gold). Rabaptin-5α is associated with clusters of vesicular tubular membranes, where it co-localized with endogenous ( A ) and overexpressed γ 1 -adaptin ( B ). The overall density and diameter of rabaptin-5α-positive membranes is reminiscent of recycling tubules. The dense cytosol between the membranes indicates the presence of high concentrations of cytosolic protein. Note that some of the membranes display a coating typical of the presence of clathrin (arrows in A and B). Double labelling of TGN46 (15 nm gold) and rabaptin-5α (10 nm gold) revealed that rabaptin-5α-positive membranes do not overlap with TGN membranes ( C ). Rabaptin-5α- (15 nm gold) positive membranes do not contain internalized BSA–5 nm gold. Arrows point to compartments that contain the endocytic marker 10 min after internalization ( D ). Double labelling of rabaptin-5α (15 nm gold) and clathrin (10 nm gold). Some of the membranes within or associated with rabaptin-5α-positive membranes also stained for clathrin (arrows). Note that the nearby endosomal vacuole (E) has a normal morphology ( E ). G = Golgi complex, L = lysosome. Bar, 200 nm.

    Article Snippet: Rabaptin constructs were cloned in pcDNA3 and pcDNA3.1His (Invitrogen, Leek, The Netherlands).

    Techniques: Transfection, Marker, Staining

    Functional cell surface expression of CD1d1 on L cells transfected with cd1d1 cDNA from murine CD1 + hematopoietic tumor cells. ( A ) Murine L cell fibroblasts were transfected with the pcDNA3.1-neo vector alone or the vector containing the full-length cd1d1 cDNA generated from L5178Y-R, YAC-1, or 18.81 cells. The cells were stained with a PE-labeled anti-mouse CD1 mAb (filled histograms). A PE-conjugated rat IgG2b (open histograms) served as an isotype control. Analysis was by cytofluorography. The data are representative of two independent experiments. ( B ) L cells transfected with vector only or vector containing the WT cd1d1 cDNA (L-CD1d1WT) or that from L5178Y-R, YAC-1, or 18.81 cells were cocultured with the Vα14 + (canonical; DN32.D3; white bars) or Vα5 + (noncanonical; N37-1A12; black bars) NKT cell hybridomas for 24 h. Supernatants were harvested, and IL-2 production was measured by ELISA. The data shown are the mean of triplicate cultures ± SD.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Inhibition of glycolipid shedding rescues recognition of a CD1+ T cell lymphoma by natural killer T (NKT) cells

    doi: 10.1073/pnas.122636199

    Figure Lengend Snippet: Functional cell surface expression of CD1d1 on L cells transfected with cd1d1 cDNA from murine CD1 + hematopoietic tumor cells. ( A ) Murine L cell fibroblasts were transfected with the pcDNA3.1-neo vector alone or the vector containing the full-length cd1d1 cDNA generated from L5178Y-R, YAC-1, or 18.81 cells. The cells were stained with a PE-labeled anti-mouse CD1 mAb (filled histograms). A PE-conjugated rat IgG2b (open histograms) served as an isotype control. Analysis was by cytofluorography. The data are representative of two independent experiments. ( B ) L cells transfected with vector only or vector containing the WT cd1d1 cDNA (L-CD1d1WT) or that from L5178Y-R, YAC-1, or 18.81 cells were cocultured with the Vα14 + (canonical; DN32.D3; white bars) or Vα5 + (noncanonical; N37-1A12; black bars) NKT cell hybridomas for 24 h. Supernatants were harvested, and IL-2 production was measured by ELISA. The data shown are the mean of triplicate cultures ± SD.

    Article Snippet: In some experiments, L cells transfected with vector alone or the WT cd1d1 cDNA in pcDNA3.1-neo (Invitrogen), generated in our laboratory as above, were used.

    Techniques: Functional Assay, Expressing, Transfection, Plasmid Preparation, Generated, Staining, Labeling, Enzyme-linked Immunosorbent Assay

    NP expression in mammalian cells leads to downreglation of PKR and eIF2α phosphorylation. HEK293T cells were transfected with NP expressing plasmid (pcDNA3.1-NP) or control plasmid (pcDNA3.1). Cells were harvested at 36 hours post-transfection and cell lysates were subjected to western blotting analysis. A. Lane 1 of panel 2 shows significant downregulation of pPKR levels in NP transfected cells. Panel 1 shows NP expression level, whereas panel 3 shows equal loading trough β-Actin control. B. Figure shows graphical representation of relative pPKR levels as measured by western blotting followed by densitometric measurement in 3 independent experiments. Error bars represent standard deviation. C. Lane 1 of panel 2 shows significant downregulation of p-eIF2α levels in NP transfected cells. Panel 1 shows NP expression level, whereas panel 3 shows equal loading trough β-Actin control. D. Figure shows graphical representation of relative p-eIF2α levels as measured by western blotting followed by densitometric measurement in 3 independent experiments. Error bars represent standard deviation.

    Journal: PLoS ONE

    Article Title: Influenza A Virus Nucleoprotein Exploits Hsp40 to Inhibit PKR Activation

    doi: 10.1371/journal.pone.0020215

    Figure Lengend Snippet: NP expression in mammalian cells leads to downreglation of PKR and eIF2α phosphorylation. HEK293T cells were transfected with NP expressing plasmid (pcDNA3.1-NP) or control plasmid (pcDNA3.1). Cells were harvested at 36 hours post-transfection and cell lysates were subjected to western blotting analysis. A. Lane 1 of panel 2 shows significant downregulation of pPKR levels in NP transfected cells. Panel 1 shows NP expression level, whereas panel 3 shows equal loading trough β-Actin control. B. Figure shows graphical representation of relative pPKR levels as measured by western blotting followed by densitometric measurement in 3 independent experiments. Error bars represent standard deviation. C. Lane 1 of panel 2 shows significant downregulation of p-eIF2α levels in NP transfected cells. Panel 1 shows NP expression level, whereas panel 3 shows equal loading trough β-Actin control. D. Figure shows graphical representation of relative p-eIF2α levels as measured by western blotting followed by densitometric measurement in 3 independent experiments. Error bars represent standard deviation.

    Article Snippet: NP gene of A/ Hatay/ 2004 (H5N1) influenza virus was cloned in pCDNA3.1 myc his vector (Invitrogen), pEGFPN1 vector (Clontech) and pGBKT7 vectors (Clontech) to generate myc-tagged, GFP-tagged and GAL4 DNA BD fused NP, respectively.

    Techniques: Expressing, Transfection, Plasmid Preparation, Western Blot, Standard Deviation

    Co-localization of IAV NP and Hsp40 in nucleus of mammalian cells. A and B. A549 cells were transfected with pcDNA3.1-NP or control pcDNA3.1 plasmid for 24 hours, and cells were fixed and processed for immunostaining. NP was stained using anti-Myc tag specific primary antibody and Alexa488 conjugated secondary antibody (Green). Hsp40 was stained using Hsp40 specific primary antibody and Alexa 594 conjugated secondary antibody (Red). Nuclei were stained with DAPI. A shows pcDNA3.1-NP transfected cells whereas B shows control pcDNA3.1 transfected cells. Panels are labeled for their respective staining. Lower right panel shows nuclear colocalization of NP and Hsp40. C and D. A549 cells were infected with PR8 influenza A virus at 1 MOI for 24 hours, and cells were fixed and processed for immunostaining. NP was stained using anti-NP monoclonal primary antibody and Alexa488 conjugated secondary antibody (Green). Hsp40 was stained using Hsp40 specific primary antibody and Alexa 594 conjugated secondary antibody (Red). Nuclei were stained with DAPI. Panels are labeled for their respective staining. C shows PR8 infected cells whereas D shows control uninfected cells. Lower right panel in C shows primarily nuclear colocalization of NP and Hsp40.

    Journal: PLoS ONE

    Article Title: Influenza A Virus Nucleoprotein Exploits Hsp40 to Inhibit PKR Activation

    doi: 10.1371/journal.pone.0020215

    Figure Lengend Snippet: Co-localization of IAV NP and Hsp40 in nucleus of mammalian cells. A and B. A549 cells were transfected with pcDNA3.1-NP or control pcDNA3.1 plasmid for 24 hours, and cells were fixed and processed for immunostaining. NP was stained using anti-Myc tag specific primary antibody and Alexa488 conjugated secondary antibody (Green). Hsp40 was stained using Hsp40 specific primary antibody and Alexa 594 conjugated secondary antibody (Red). Nuclei were stained with DAPI. A shows pcDNA3.1-NP transfected cells whereas B shows control pcDNA3.1 transfected cells. Panels are labeled for their respective staining. Lower right panel shows nuclear colocalization of NP and Hsp40. C and D. A549 cells were infected with PR8 influenza A virus at 1 MOI for 24 hours, and cells were fixed and processed for immunostaining. NP was stained using anti-NP monoclonal primary antibody and Alexa488 conjugated secondary antibody (Green). Hsp40 was stained using Hsp40 specific primary antibody and Alexa 594 conjugated secondary antibody (Red). Nuclei were stained with DAPI. Panels are labeled for their respective staining. C shows PR8 infected cells whereas D shows control uninfected cells. Lower right panel in C shows primarily nuclear colocalization of NP and Hsp40.

    Article Snippet: NP gene of A/ Hatay/ 2004 (H5N1) influenza virus was cloned in pCDNA3.1 myc his vector (Invitrogen), pEGFPN1 vector (Clontech) and pGBKT7 vectors (Clontech) to generate myc-tagged, GFP-tagged and GAL4 DNA BD fused NP, respectively.

    Techniques: Transfection, Plasmid Preparation, Immunostaining, Staining, Labeling, Infection

    Detection of IAV NP-Hsp40 interaction in mammalian cells tranfected with NP expressing plasmid by co-immunoprecipitation. A. HEK293T cells were pcDNA3.1-NP and pcDNA3.1-Hsp40 plasmids alone or in combination, followed by metabolic labeling with S 35 . 48 hours post-transfection cells were harvested and IP was setup using anti-NP-specific antibody and anti-Hsp40-specific antibody followed by autoradiography. Lanes 2 and 4 show co-IP of Hsp40 with NP and vice-versa. Lanes 1 and 3 show anti-NP and anti-Hsp40 antibodies were not cross reacting with Hsp40 and NP, respectively. B. A549 cells were transfected with pcDNA3.1-NP plasmid or control pcDNA3.1 plasmid. Cells were harvested 48 hours post-transfection and immunoprecipitation was setup using anti-Myc tag antibody and anti-Hsp40 antibody, followed by western blotting. Lane 2 of panel 1 shows co-IP of NP with Hsp40 and lane 2 of panel 2 shows co-IP Hsp40 with NP. Lane 1 of panel 1 and 2 represents control samples transfected with empty vector. Panels 3 and 4 show expression levels of NP and Hsp40 in cell lysates.

    Journal: PLoS ONE

    Article Title: Influenza A Virus Nucleoprotein Exploits Hsp40 to Inhibit PKR Activation

    doi: 10.1371/journal.pone.0020215

    Figure Lengend Snippet: Detection of IAV NP-Hsp40 interaction in mammalian cells tranfected with NP expressing plasmid by co-immunoprecipitation. A. HEK293T cells were pcDNA3.1-NP and pcDNA3.1-Hsp40 plasmids alone or in combination, followed by metabolic labeling with S 35 . 48 hours post-transfection cells were harvested and IP was setup using anti-NP-specific antibody and anti-Hsp40-specific antibody followed by autoradiography. Lanes 2 and 4 show co-IP of Hsp40 with NP and vice-versa. Lanes 1 and 3 show anti-NP and anti-Hsp40 antibodies were not cross reacting with Hsp40 and NP, respectively. B. A549 cells were transfected with pcDNA3.1-NP plasmid or control pcDNA3.1 plasmid. Cells were harvested 48 hours post-transfection and immunoprecipitation was setup using anti-Myc tag antibody and anti-Hsp40 antibody, followed by western blotting. Lane 2 of panel 1 shows co-IP of NP with Hsp40 and lane 2 of panel 2 shows co-IP Hsp40 with NP. Lane 1 of panel 1 and 2 represents control samples transfected with empty vector. Panels 3 and 4 show expression levels of NP and Hsp40 in cell lysates.

    Article Snippet: NP gene of A/ Hatay/ 2004 (H5N1) influenza virus was cloned in pCDNA3.1 myc his vector (Invitrogen), pEGFPN1 vector (Clontech) and pGBKT7 vectors (Clontech) to generate myc-tagged, GFP-tagged and GAL4 DNA BD fused NP, respectively.

    Techniques: Expressing, Plasmid Preparation, Immunoprecipitation, Labeling, Transfection, Autoradiography, Co-Immunoprecipitation Assay, Western Blot

    Localized gene delivery mediated by GAC. X-gal staining of pcDNA3.1-LacZ transfected COS7 cells appeared blue in (A) complete view and (B) enlarged view. Transfected COS7 cells locally expressed EGFP in designed areas. (C) Fluorescence microscopic picture, (D) phase-contrast microscopic picture and (E) merged picture. The dotted lines indicate the border of the GAC-coated and uncoated areas. (Bar in A = 5 mm; Bars in B–E = 100 μm).

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Localized SDF-1alpha gene release mediated by collagen substrate induces CD117+ stem cells homing

    doi: 10.1111/j.1582-4934.2008.00624.x

    Figure Lengend Snippet: Localized gene delivery mediated by GAC. X-gal staining of pcDNA3.1-LacZ transfected COS7 cells appeared blue in (A) complete view and (B) enlarged view. Transfected COS7 cells locally expressed EGFP in designed areas. (C) Fluorescence microscopic picture, (D) phase-contrast microscopic picture and (E) merged picture. The dotted lines indicate the border of the GAC-coated and uncoated areas. (Bar in A = 5 mm; Bars in B–E = 100 μm).

    Article Snippet: Amplification and purification of plasmid DNA Plasmid pRE-Luciferase (pREP4-Luc) and pcDNA3.1-LacZ (Invitrogen, Carlsbad, CA, USA) were used as received without further modification.

    Techniques: Staining, Transfection, Fluorescence

    Effects of RE-IIBP transcript by RNA interference. (A) HeLa cells were mock transfected or transfected with pcDNA3.1-HisTOPO-RE-IIBP and pSM2c-WHSC1/MMSET-shRNA, and WHSC1/MMESET and RE-IIBP expression levels were confirmed by RT-PCR with specific primers.

    Journal:

    Article Title: Multiple Myeloma-Related WHSC1/MMSET Isoform RE-IIBP Is a Histone Methyltransferase with Transcriptional Repression Activity ▿

    doi: 10.1128/MCB.02130-07

    Figure Lengend Snippet: Effects of RE-IIBP transcript by RNA interference. (A) HeLa cells were mock transfected or transfected with pcDNA3.1-HisTOPO-RE-IIBP and pSM2c-WHSC1/MMSET-shRNA, and WHSC1/MMESET and RE-IIBP expression levels were confirmed by RT-PCR with specific primers.

    Article Snippet: For the construction of mammalian expression vectors, we employed pcDNA3.1-HisTOPO (Invitrogen) and CMX-Gal4 in order to create the His-tagged RE-IIBP and Gal4-RE-IIBP proteins.

    Techniques: Transfection, shRNA, Expressing, Reverse Transcription Polymerase Chain Reaction

    RE-IIBP represses transcription and associates with HDAC. (A) HeLa cells were transfected with Gal4-SV40 and increasing concentrations of RE-IIBP constructs, as indicated. Following transfection of the pcDNA3.1-HisTOPO-RE-IIBP and the point mutant (C483A),

    Journal:

    Article Title: Multiple Myeloma-Related WHSC1/MMSET Isoform RE-IIBP Is a Histone Methyltransferase with Transcriptional Repression Activity ▿

    doi: 10.1128/MCB.02130-07

    Figure Lengend Snippet: RE-IIBP represses transcription and associates with HDAC. (A) HeLa cells were transfected with Gal4-SV40 and increasing concentrations of RE-IIBP constructs, as indicated. Following transfection of the pcDNA3.1-HisTOPO-RE-IIBP and the point mutant (C483A),

    Article Snippet: For the construction of mammalian expression vectors, we employed pcDNA3.1-HisTOPO (Invitrogen) and CMX-Gal4 in order to create the His-tagged RE-IIBP and Gal4-RE-IIBP proteins.

    Techniques: Transfection, Construct, Mutagenesis

    Rep and Cap, but not ORF3, induced unfolded protein response via PERK activation PK-15 cells were transfected with the indicated plasmids (p-Flag stands for pcDNA3.1-Flag, p-EGFP for pcDNA3.1-EGFP, p-ORF3-EGFP for pcDNA3.1-ORF3-EGFP, p-Rep for p-Rep-Flag, p-Cap and p-Cap-Flag, the same for the following figures) for 12, 24, 36, and 48 h. Western blotting was performed to visualize phosphorylated forms of PERK (p-PERK) and eIF2α (p-eIF2α), total PERK (t-PERK) and total eIF2α (t-eIF2α), and ER stress marker GRP78 in lysates of cells expressing Rep or Cap (a) and of those expressing ORF3 (b). Expressions of the fusion proteins Cap, Rep, and ORF3 were revealed by antibodies to Flag or EGFP

    Journal: Journal of Zhejiang University. Science. B

    Article Title: Porcine circovirus type 2 capsid protein induces unfolded protein response with subsequent activation of apoptosis *

    doi: 10.1631/jzus.B1600208

    Figure Lengend Snippet: Rep and Cap, but not ORF3, induced unfolded protein response via PERK activation PK-15 cells were transfected with the indicated plasmids (p-Flag stands for pcDNA3.1-Flag, p-EGFP for pcDNA3.1-EGFP, p-ORF3-EGFP for pcDNA3.1-ORF3-EGFP, p-Rep for p-Rep-Flag, p-Cap and p-Cap-Flag, the same for the following figures) for 12, 24, 36, and 48 h. Western blotting was performed to visualize phosphorylated forms of PERK (p-PERK) and eIF2α (p-eIF2α), total PERK (t-PERK) and total eIF2α (t-eIF2α), and ER stress marker GRP78 in lysates of cells expressing Rep or Cap (a) and of those expressing ORF3 (b). Expressions of the fusion proteins Cap, Rep, and ORF3 were revealed by antibodies to Flag or EGFP

    Article Snippet: For the construction of p-Rep-Flag and p-Cap-Flag, the orf1 and orf2 genes were amplified from the genomic DNA of the PCV2 and subcloned into pcDNA3.1-Flag (Invitrogen).

    Techniques: Activation Assay, Transfection, Western Blot, Marker, Expressing

    The suppression of BAP31 expression induces autophagy and antagonizes ER stress-induced cell death. ( a ) Loss of BAP31 increases LC3-II expression. U2OS cells were transfected with 150 pmol of siBAP31 or siControl for 24 h. Cells were subjected to immunoblotting using anti-BAP31, anti-LC3, and anti-β-actin antibodies. ( b ) U2OS cells stably expressing GFP-LC3 were transfected with 150 pmol of siBAP31 or siControl for 24 h. Cells were fixed with 4% PFA, and GFP-LC3 (green) fluorescence was determined. Blue represents nuclear DAPI staining. Scale bar, 10 μm. ( c ) U2OS cells were transfected with siBAP31 (+) or siControl (−) for 18 h and then transfected with HA-BAP31 (+) or pcDNA3.1 (−) for 12 h. Cells were subjected to immunoblotting using indicated antibodies. ( d ) BAP31 knockdown stimulates autophagosome synthesis. U2OS cells were transfected with 150 pmol of siBAP31 or siControl for 24 h, followed by treatment with or without 1 µg/mL of bafilomycin A1 for 1 h. Cells were subjected to immunoblotting using the indicated antibodies. ( e , f ) The suppression of BAP31 inhibits ER stress-mediated cell death by inducting autophagy. The cells were transfected with siBAP31 or siControl for 16 h and these cells were preincubated with or withou t 5 mM of 3-MA for 1 h and further incubated with or without BFA (1 µg/mL) for 18 h in U2OS, HeLa, and MEF cells. Cells were subjected to immunoblotting using the indicated antibodies ( e ) or MTT using cell viability assay ( f ). These experiments were repeated two times ( a – e ). Data are presented as the mean ± standard deviation (SD) of the three simultaneously performed experiments ( f ). P values were calculated using two-way ANOVA; n.s., not significant; ** P

    Journal: Cells

    Article Title: BAP31 Inhibits Cell Adaptation to ER Stress Conditions, Negatively Regulating Autophagy Induction by Interaction with STX17

    doi: 10.3390/cells8111350

    Figure Lengend Snippet: The suppression of BAP31 expression induces autophagy and antagonizes ER stress-induced cell death. ( a ) Loss of BAP31 increases LC3-II expression. U2OS cells were transfected with 150 pmol of siBAP31 or siControl for 24 h. Cells were subjected to immunoblotting using anti-BAP31, anti-LC3, and anti-β-actin antibodies. ( b ) U2OS cells stably expressing GFP-LC3 were transfected with 150 pmol of siBAP31 or siControl for 24 h. Cells were fixed with 4% PFA, and GFP-LC3 (green) fluorescence was determined. Blue represents nuclear DAPI staining. Scale bar, 10 μm. ( c ) U2OS cells were transfected with siBAP31 (+) or siControl (−) for 18 h and then transfected with HA-BAP31 (+) or pcDNA3.1 (−) for 12 h. Cells were subjected to immunoblotting using indicated antibodies. ( d ) BAP31 knockdown stimulates autophagosome synthesis. U2OS cells were transfected with 150 pmol of siBAP31 or siControl for 24 h, followed by treatment with or without 1 µg/mL of bafilomycin A1 for 1 h. Cells were subjected to immunoblotting using the indicated antibodies. ( e , f ) The suppression of BAP31 inhibits ER stress-mediated cell death by inducting autophagy. The cells were transfected with siBAP31 or siControl for 16 h and these cells were preincubated with or withou t 5 mM of 3-MA for 1 h and further incubated with or without BFA (1 µg/mL) for 18 h in U2OS, HeLa, and MEF cells. Cells were subjected to immunoblotting using the indicated antibodies ( e ) or MTT using cell viability assay ( f ). These experiments were repeated two times ( a – e ). Data are presented as the mean ± standard deviation (SD) of the three simultaneously performed experiments ( f ). P values were calculated using two-way ANOVA; n.s., not significant; ** P

    Article Snippet: To generate stable cell lines expressing GFP-LC3 (pEGFP-LC3; Addgene #21073, Cambridge, MA, USA) or transiently expressed Flag-STX17 (Addgene #45911, Cambridge, MA, USA), pcDNA3.1 (control) (invitrogen-Thermo Fisher Scientific, Hampton, NH, USA), HA-BAP31 [ ], HA-ATG14L (Addgene #24294, Cambridge, MA, USA) or DsRed-ER (Takara Bio Inc, Tokyo, Japan) constructs were introduced into the U2OS cells using lipofection methods.

    Techniques: Expressing, Transfection, Stable Transfection, Fluorescence, Staining, Incubation, MTT Assay, Viability Assay, Standard Deviation

    ATF4 induced CSE transcription via direct binding to a cis regulatory intronic site. A and B , luciferase activity resulting from HEK cells transfected with constructs as indicated together with (empty vector control plasmid) pCDNA3.1 or overexpressed ATF4. RLU , relative light units. n = 4. *, p

    Journal: The Journal of Biological Chemistry

    Article Title: Transcriptional Regulation of Cystathionine-γ-Lyase in Endothelial Cells by NADPH Oxidase 4-Dependent Signaling *

    doi: 10.1074/jbc.M115.685578

    Figure Lengend Snippet: ATF4 induced CSE transcription via direct binding to a cis regulatory intronic site. A and B , luciferase activity resulting from HEK cells transfected with constructs as indicated together with (empty vector control plasmid) pCDNA3.1 or overexpressed ATF4. RLU , relative light units. n = 4. *, p

    Article Snippet: Transient transfections of (control) pCDNA3.1 (Invitrogen) or an equivalent plasmid in which the full-length mouse ATF4 cDNA had been inserted (pATF4; Addgene) were carried out using the turbofect reagent (Thermo Scientific) following the manufacturers' specifications using 4 μg of plasmid DNA/6-cm dish and incubated for 24 h. For silencing experiments, HUVECs were cultured in 6-cm dishes and serum-starved for 1 h in Opti-MEM before being transfected with siRNAs targeted to Nox4 (5 nm ; s27015), p22Phox (20 nm ; s194371), ATF4 (20 nm ; s1702), heme-regulated inhibitor kinase (HRI; 20 nm ; s25823), protein kinase R-like ER kinase (PERK; 20 nm ; s18101), or scrambled control siRNA at equivalent concentrations (all Ambion Silencer-select) and incubated for 24 or 48 h as indicated.

    Techniques: Binding Assay, Luciferase, Activity Assay, Transfection, Construct, Plasmid Preparation

    Nox4 regulated CSE expression via ATF4. A , representative Western blot and quantitative densitometric analyses of ATF4 protein expression and the corresponding QPCR analyses of CSE mRNA expression in HUVECs after 24-h ATF4 or control pCDNA3.1 overexpression. A.U. , absorbance units. B , representative Western blot and quantitative densitometric analyses of ATF4 protein expression in HUVEC after 48 h of treatment with ATF4-targeted siRNA (siATF4) or control siRNA (siScram) together with 24 h Nox4 or β-gal ( B Gal ) overexpression as indicated. C , QPCR analyses of ATF4 and CSE mRNA expression in HUVECs after treatments as in B. D , representative Western blot and corresponding densitometric analyses of ATF4 protein expression in CMECs isolated from WT and eNox4 Tg mice. All data are normalized to β-actin mRNA and protein expression. n = 3; *, p

    Journal: The Journal of Biological Chemistry

    Article Title: Transcriptional Regulation of Cystathionine-γ-Lyase in Endothelial Cells by NADPH Oxidase 4-Dependent Signaling *

    doi: 10.1074/jbc.M115.685578

    Figure Lengend Snippet: Nox4 regulated CSE expression via ATF4. A , representative Western blot and quantitative densitometric analyses of ATF4 protein expression and the corresponding QPCR analyses of CSE mRNA expression in HUVECs after 24-h ATF4 or control pCDNA3.1 overexpression. A.U. , absorbance units. B , representative Western blot and quantitative densitometric analyses of ATF4 protein expression in HUVEC after 48 h of treatment with ATF4-targeted siRNA (siATF4) or control siRNA (siScram) together with 24 h Nox4 or β-gal ( B Gal ) overexpression as indicated. C , QPCR analyses of ATF4 and CSE mRNA expression in HUVECs after treatments as in B. D , representative Western blot and corresponding densitometric analyses of ATF4 protein expression in CMECs isolated from WT and eNox4 Tg mice. All data are normalized to β-actin mRNA and protein expression. n = 3; *, p

    Article Snippet: Transient transfections of (control) pCDNA3.1 (Invitrogen) or an equivalent plasmid in which the full-length mouse ATF4 cDNA had been inserted (pATF4; Addgene) were carried out using the turbofect reagent (Thermo Scientific) following the manufacturers' specifications using 4 μg of plasmid DNA/6-cm dish and incubated for 24 h. For silencing experiments, HUVECs were cultured in 6-cm dishes and serum-starved for 1 h in Opti-MEM before being transfected with siRNAs targeted to Nox4 (5 nm ; s27015), p22Phox (20 nm ; s194371), ATF4 (20 nm ; s1702), heme-regulated inhibitor kinase (HRI; 20 nm ; s25823), protein kinase R-like ER kinase (PERK; 20 nm ; s18101), or scrambled control siRNA at equivalent concentrations (all Ambion Silencer-select) and incubated for 24 or 48 h as indicated.

    Techniques: Expressing, Western Blot, Real-time Polymerase Chain Reaction, Over Expression, Isolation, Mouse Assay

    PHB1 overcomes the anti-proliferative effect of miRNA-195. ( a ) Proliferation assay based on nuclear counting per mm 2 . UACC-62 melanoma cells were transfected with either miR-control or miR-195 (25 nM) and observed for five days after transfection. ( b ) To conduct rescue experiments, UACC-62 melanoma cells were stably expressing either ORF-PHB1 or pcDNA3.1-EV. Cells were then transfected with either miRNA-mimics control or miR-195 mimics. After transfection, the proliferation rate was measured for six days and the results showed that cells transfected with transgenic PHB1 overcome the suppressive effect of miR-195 (green line) compared to pcDNA3.1-EV cells (pink line). Representative examples of at least three independent experiments are reported

    Journal: BMC Cancer

    Article Title: MicroRNA-195 acts as an anti-proliferative miRNA in human melanoma cells by targeting Prohibitin 1

    doi: 10.1186/s12885-017-3721-7

    Figure Lengend Snippet: PHB1 overcomes the anti-proliferative effect of miRNA-195. ( a ) Proliferation assay based on nuclear counting per mm 2 . UACC-62 melanoma cells were transfected with either miR-control or miR-195 (25 nM) and observed for five days after transfection. ( b ) To conduct rescue experiments, UACC-62 melanoma cells were stably expressing either ORF-PHB1 or pcDNA3.1-EV. Cells were then transfected with either miRNA-mimics control or miR-195 mimics. After transfection, the proliferation rate was measured for six days and the results showed that cells transfected with transgenic PHB1 overcome the suppressive effect of miR-195 (green line) compared to pcDNA3.1-EV cells (pink line). Representative examples of at least three independent experiments are reported

    Article Snippet: Stable cell lines generation UACC-62 cells stably expressing PHB1-ORF (Open Reading Frame, without 5′ and 3’UTR) or pcDNA3.1-EV (empty vector) (Invitrogen/Thermo Fisher Scientific, Waltham, MA, USA) were generated by transfection followed by G418 selection (Gibco/Thermo Fisher Scientific, Waltham, MA, USA) (0.8 mg/mL).

    Techniques: Proliferation Assay, Transfection, Stable Transfection, Expressing, Transgenic Assay

    Detection of intracellular CD83 in monocytes, macrophages and imDCs ( A ) Monocytes (Mo), macrophages (MΦ) and imDCs were fixed with paraformaldehyde and permeabilized with saponin. Upon blocking with 20% (v/v) goat serum, the cells were stained with a PE-labelled CD83 antibody (clone HB15e; filled histograms) or, as a control, isotype IgG (open histograms). The monocytic THP-1 cells were used as controls. ( B ) Monocytes (lane 3), resting macrophages (lane 4), LPS-activated macrophages (lane 5), imDCs (lane 6) and mDCs (lane 7) were lysed with Nonidet P40, and the soluble fractions were analysed by Western blotting using the HB15a CD83 antibody (Santa Cruz Biotechnology). Aliquots (50 μg) of each sample were separated by SDS/PAGE. Also included in the blots was the soluble fraction of THP-1 cells (lane 8), and 293T cells transfected with the pcDNA3.1 plasmid (lane 1) or the phCD83 expression vector (lane 2). The transfected cells were cultured for 24 h. For monocytes, both the soluble (lane 9) and insoluble (lane 10) fractions were subjected to Western blot analysis. ( C ) Expression of CD83 by 293T cells: 293T cells were transfected with the phCD83 expression vector (lower panel) or, as a control, with the pcDNA3.1 plasmid (upper panel). The cells were stained with a PE-labelled CD83 antibody (HB15e; open histograms). The filled histograms represent signals detected with isotype IgG. All results shown are representative of at least three similar experiments.

    Journal: Biochemical Journal

    Article Title: CD83 is preformed inside monocytes, macrophages and dendritic cells, but it is only stably expressed on activated dendritic cells

    doi: 10.1042/BJ20040741

    Figure Lengend Snippet: Detection of intracellular CD83 in monocytes, macrophages and imDCs ( A ) Monocytes (Mo), macrophages (MΦ) and imDCs were fixed with paraformaldehyde and permeabilized with saponin. Upon blocking with 20% (v/v) goat serum, the cells were stained with a PE-labelled CD83 antibody (clone HB15e; filled histograms) or, as a control, isotype IgG (open histograms). The monocytic THP-1 cells were used as controls. ( B ) Monocytes (lane 3), resting macrophages (lane 4), LPS-activated macrophages (lane 5), imDCs (lane 6) and mDCs (lane 7) were lysed with Nonidet P40, and the soluble fractions were analysed by Western blotting using the HB15a CD83 antibody (Santa Cruz Biotechnology). Aliquots (50 μg) of each sample were separated by SDS/PAGE. Also included in the blots was the soluble fraction of THP-1 cells (lane 8), and 293T cells transfected with the pcDNA3.1 plasmid (lane 1) or the phCD83 expression vector (lane 2). The transfected cells were cultured for 24 h. For monocytes, both the soluble (lane 9) and insoluble (lane 10) fractions were subjected to Western blot analysis. ( C ) Expression of CD83 by 293T cells: 293T cells were transfected with the phCD83 expression vector (lower panel) or, as a control, with the pcDNA3.1 plasmid (upper panel). The cells were stained with a PE-labelled CD83 antibody (HB15e; open histograms). The filled histograms represent signals detected with isotype IgG. All results shown are representative of at least three similar experiments.

    Article Snippet: The PCR product was cloned into the Kpn I/ Apa I site of the pcDNA3.1 plasmid (Invitrogen) to generate the phCD83 expression vector.

    Techniques: Blocking Assay, Staining, Western Blot, SDS Page, Transfection, Plasmid Preparation, Expressing, Cell Culture

    CFIm25 overexpression results in 3′-UTR lengthening and decreased protein expression of target genes. Primary fibroblasts isolated from healthy and IPF lungs were electroporated with empty or CFIm25-overexpressing pCDNA3.1 plasmids. Two or three days after transfection, cells were collected for ( A ) Western blotting to determine the protein levels of CFIm25 and its target genes and ( B ) qRT-PCR to determine the dPAS usage of the CFIm25 targets COL1A1, TGFBR1, WNT5A , and FZD2 . n = 3 biological replicates. * P

    Journal: The Journal of Clinical Investigation

    Article Title: Cleavage factor 25 deregulation contributes to pulmonary fibrosis through alternative polyadenylation

    doi: 10.1172/JCI122106

    Figure Lengend Snippet: CFIm25 overexpression results in 3′-UTR lengthening and decreased protein expression of target genes. Primary fibroblasts isolated from healthy and IPF lungs were electroporated with empty or CFIm25-overexpressing pCDNA3.1 plasmids. Two or three days after transfection, cells were collected for ( A ) Western blotting to determine the protein levels of CFIm25 and its target genes and ( B ) qRT-PCR to determine the dPAS usage of the CFIm25 targets COL1A1, TGFBR1, WNT5A , and FZD2 . n = 3 biological replicates. * P

    Article Snippet: For CFIm25 overexpression, the coding region (CDS) of the human CFIm25 gene was cloned into pCDNA3.1 plasmids (Thermo Fisher Scientific).

    Techniques: Over Expression, Expressing, Isolation, Transfection, Western Blot, Quantitative RT-PCR

    miR-144-3p mediated downregulation of ERO1L led to suppression of STAT3 activity. (A) KB (left panels) and CAL-27 (right panels) cells were transfected with the miR-144-3p mimics or negative control (NC). (B) HOEC cells were transfected with the miR-144-3p inhibitor or negative control (NC-inhibitor). (C) LV-miR-144-3p lentiviruses infected KB (left panels) and CAL-27 (right panels) cells were transfected with pcDNA3.1-ERO1L or the empty vector pcDNA3.1 (V) for 48 h. (D) miR-144-3p inhibitor transduced HOEC cells were transfected with the control siRNAs (siNC) or siRNAs targeting ERO1L for 48 h. (A-D) Cell lysates were subjected to immunoblotting with the indicated antibodies (upper panels). The relative quantitation of p-STAT3 determined by scanning densitometry analysis upon normalization for STAT3 (lower panels). (E) Tumor tissues derived from CAL-27 cells transduced with LV-miR-144-3p or LV lentiviruses were subjected to immunoblotting with the indicated antibodies (left panel) and densitometric analysis of this experiment normalized to STAT3 is shown (right panel). Data indicate mean ± SD of triplicate samples from one of three representative experiments. * P

    Journal: Journal of Cancer

    Article Title: MicroRNA-144-3p Inhibits Tumorigenesis of Oral Squamous Cell Carcinoma by downregulating ERO1L

    doi: 10.7150/jca.33267

    Figure Lengend Snippet: miR-144-3p mediated downregulation of ERO1L led to suppression of STAT3 activity. (A) KB (left panels) and CAL-27 (right panels) cells were transfected with the miR-144-3p mimics or negative control (NC). (B) HOEC cells were transfected with the miR-144-3p inhibitor or negative control (NC-inhibitor). (C) LV-miR-144-3p lentiviruses infected KB (left panels) and CAL-27 (right panels) cells were transfected with pcDNA3.1-ERO1L or the empty vector pcDNA3.1 (V) for 48 h. (D) miR-144-3p inhibitor transduced HOEC cells were transfected with the control siRNAs (siNC) or siRNAs targeting ERO1L for 48 h. (A-D) Cell lysates were subjected to immunoblotting with the indicated antibodies (upper panels). The relative quantitation of p-STAT3 determined by scanning densitometry analysis upon normalization for STAT3 (lower panels). (E) Tumor tissues derived from CAL-27 cells transduced with LV-miR-144-3p or LV lentiviruses were subjected to immunoblotting with the indicated antibodies (left panel) and densitometric analysis of this experiment normalized to STAT3 is shown (right panel). Data indicate mean ± SD of triplicate samples from one of three representative experiments. * P

    Article Snippet: The ERO1L overexpression plasmid was constructed by introducing the human ERO1L cDNA into the pcDNA3.1 vector (Invitrogen), and was designated as pcDNA3.1-ERO1L.

    Techniques: Activity Assay, Transfection, Negative Control, Infection, Plasmid Preparation, Quantitation Assay, Derivative Assay, Transduction

    (A) Vpr is coprecipitated with p300 from HeLa cell extracts. Coprecipitation is suppressed by E1A or p300(2045-2191). HeLa cells were transfected with pFLAG-CMV2 (lane 2) or pCMV-FLAG-VPR (lanes 3, 4, 5, and 6). pCMV-12S-E1A or pCDNA3.1-His/A-p300(2045-2191) was cotransfected in lanes 5 and 6, respectively. Vpr-p300 complexes were precipitated with anti-human p300 antibody and separated on a 14% SDS-PAGE gel. To determine the levels of expressed Vpr protein, Vpr was precipitated with anti-FLAG antibody from the same sample and separated on a 14% SDS-PAGE gel. Proteins were transferred to membranes and probed with anti-FLAG (M2) antibody. Five percent of the total lysate from cells transfected with pCMV-FLAG-VPR was run as a marker (lane 1). (B) His-p300(2045-2191) and GAL4-p300(1945-2414) are expressed in HeLa cells. HeLa cells were transfected with pCDNA3.1-His/A or pCDNA3-His/A-p300(2045-2191) or with pCDNA3 or pCDNA3-GAL4-p300(1945-2414), and cell lysates were analyzed on 14% or 10% SDS-PAGE gels, respectively. Transferred protein was blotted with anti-His or anti-GAL4 antibody. (C) Vpr forms a complex with p300 and GR in the presence of dexamethasone. HeLa cells were transfected with pFLAG-CMV2 (lanes 2 and 3) or pCMV-FLAG-VPR (lanes 4 and 5). Dexamethasone (10 −5 M) was added to the medium 5 h before harvest. The Vpr/p300/GR complex was precipitated with anti-human p300 antibody or anti-GR antibody and separated on 14% or 8% SDS-PAGE gels, respectively. Proteins were transferred to membranes and probed with anti-FLAG(M2) antibody (gels a and b) or anti-GR antibody (gel c). Five percent of the total lysate from cells transfected with pCMV-FLAG-VPR was run as a marker (lane 1). To determine the levels of expressed Vpr, p300, or GR proteins, the same amounts of cell lysates were separated on 14%, 6%, or 8% SDS-PAGE gels and blotted with anti-FLAG(M2), anti-p300, or anti-GR antibody, respectively (gels d, e, and f).

    Journal: Journal of Virology

    Article Title: Human Immunodeficiency Virus Type 1 (HIV-1) Accessory Protein Vpr Induces Transcription of the HIV-1 and Glucocorticoid-Responsive Promoters by Binding Directly to p300/CBP Coactivators

    doi: 10.1128/JVI.76.19.9724-9734.2002

    Figure Lengend Snippet: (A) Vpr is coprecipitated with p300 from HeLa cell extracts. Coprecipitation is suppressed by E1A or p300(2045-2191). HeLa cells were transfected with pFLAG-CMV2 (lane 2) or pCMV-FLAG-VPR (lanes 3, 4, 5, and 6). pCMV-12S-E1A or pCDNA3.1-His/A-p300(2045-2191) was cotransfected in lanes 5 and 6, respectively. Vpr-p300 complexes were precipitated with anti-human p300 antibody and separated on a 14% SDS-PAGE gel. To determine the levels of expressed Vpr protein, Vpr was precipitated with anti-FLAG antibody from the same sample and separated on a 14% SDS-PAGE gel. Proteins were transferred to membranes and probed with anti-FLAG (M2) antibody. Five percent of the total lysate from cells transfected with pCMV-FLAG-VPR was run as a marker (lane 1). (B) His-p300(2045-2191) and GAL4-p300(1945-2414) are expressed in HeLa cells. HeLa cells were transfected with pCDNA3.1-His/A or pCDNA3-His/A-p300(2045-2191) or with pCDNA3 or pCDNA3-GAL4-p300(1945-2414), and cell lysates were analyzed on 14% or 10% SDS-PAGE gels, respectively. Transferred protein was blotted with anti-His or anti-GAL4 antibody. (C) Vpr forms a complex with p300 and GR in the presence of dexamethasone. HeLa cells were transfected with pFLAG-CMV2 (lanes 2 and 3) or pCMV-FLAG-VPR (lanes 4 and 5). Dexamethasone (10 −5 M) was added to the medium 5 h before harvest. The Vpr/p300/GR complex was precipitated with anti-human p300 antibody or anti-GR antibody and separated on 14% or 8% SDS-PAGE gels, respectively. Proteins were transferred to membranes and probed with anti-FLAG(M2) antibody (gels a and b) or anti-GR antibody (gel c). Five percent of the total lysate from cells transfected with pCMV-FLAG-VPR was run as a marker (lane 1). To determine the levels of expressed Vpr, p300, or GR proteins, the same amounts of cell lysates were separated on 14%, 6%, or 8% SDS-PAGE gels and blotted with anti-FLAG(M2), anti-p300, or anti-GR antibody, respectively (gels d, e, and f).

    Article Snippet: Expression vectors for GST-p300(1925-2191), GST-p300(2184-2414), GST-p300(1925-2045), and GST-p300(2045-2191) were constructed by introducing PCR fragments of corresponding portions of p300 cDNA, amplified from pCDNA3-GAL4-p300(1-2414) (a gift from A. Giordano, Jefferson Medical College, Philadelphia, Pa.), into pGEX-4T3 (Amersham Pharmacia Biotech, Piscataway, N.J.). pGEX-4T3-GRα, which expresses GST-GRα fusion protein, was constructed by inserting the human GRα coding region into pGEX-4T3. pCDNA3.1-His/A-p300(2045-2191) was constructed by subcloning a PCR-amplified cDNA fragment corresponding to p300(2045-2191) from pCDNA3-GAL4-p300(1-2414) into pCDNA3.1-His/A (Invitrogen). pM-SRC-1a was constructed by subcloning SRC-1a cDNA from pCR3.1-SRC1a (a gift from B. W. O'Malley, Baylor College of Medicine, Houston, Tex.) into pM (Clontech).

    Techniques: Transfection, SDS Page, Marker

    EGF induces the colocalization of AMAP1 with PRKD2 via Rab5c. (A–F) MDA-MB-231 cells were cultured on collagen I–coated dishes, then transfected with Rab5c siRNA and pCMV-V5-PRKD2 one after another (A–C) or with pCMV-V5-PRKD2 and pcDNA3.1 HisC Rab5c simultaneously. Cells were serum-starved for 2 h, then treated (A and D) or left untreated (B and E) with 10 ng/ml EGF (5 min) before fixation. AMAP1, Xpress, and V5 epitopes were immunostained. In the merged image, V5-PRKD2 and AMAP1 are shown as green and red, respectively, in A and B; V5-PRKD2, Xpress-Rab5c, and AMAP1 are shown as green, red, and blue, respectively, in D and E. Colocalization indices of each protein were quantified, and the results represent mean ±SEM of > 10 measurements (error bars; C and F). The rightmost panels in D and E show enlarged views of the boxed regions. Bars, 10 µm.

    Journal: The Journal of Cell Biology

    Article Title: Rab5c promotes AMAP1-PRKD2 complex formation to enhance ?1 integrin recycling in EGF-induced cancer invasion

    doi: 10.1083/jcb.201201065

    Figure Lengend Snippet: EGF induces the colocalization of AMAP1 with PRKD2 via Rab5c. (A–F) MDA-MB-231 cells were cultured on collagen I–coated dishes, then transfected with Rab5c siRNA and pCMV-V5-PRKD2 one after another (A–C) or with pCMV-V5-PRKD2 and pcDNA3.1 HisC Rab5c simultaneously. Cells were serum-starved for 2 h, then treated (A and D) or left untreated (B and E) with 10 ng/ml EGF (5 min) before fixation. AMAP1, Xpress, and V5 epitopes were immunostained. In the merged image, V5-PRKD2 and AMAP1 are shown as green and red, respectively, in A and B; V5-PRKD2, Xpress-Rab5c, and AMAP1 are shown as green, red, and blue, respectively, in D and E. Colocalization indices of each protein were quantified, and the results represent mean ±SEM of > 10 measurements (error bars; C and F). The rightmost panels in D and E show enlarged views of the boxed regions. Bars, 10 µm.

    Article Snippet: cDNAs and transfection AMAP1 cDNA ( ) was cloned into pEGFP C1 (Takara Bio Inc.). cDNA for PRKD2, amplified by PCR from the first-strand cDNA of MDA-MB-231 cells, was cloned into pmVenus C1 (see below), pcDNA3.1 HisC (Invitrogen), or pCMV-V5 (see below).

    Techniques: Multiple Displacement Amplification, Cell Culture, Transfection

    Association of PRKD2 with AMAP1 in highly invasive breast cancer cells. (A) Lysates from different breast cancer cells and HMECs were probed with the indicated antibodies. (B) Lysates from the indicated cells were immunoprecipitated with an anti-AMAP1 antibody or an anti-AMAP2 antibody, then subjected to blotting with the indicated antibodies. Preimmune serum of the anti-AMAP1 antibody was used as a control (Pre-imm). WCL, whole cell lysate (5 µg). (C and D) MDA-MB-231 cells, expressing mVenus-PKD2 and cultured on Alexa Fluor–labeled gelatin film, were fixed and stained with an anti-AMAP1 antibody. Original colors were: PRKD2, green; AMAP1, red; and sites of gelatin degradation, blue (C). Bar, 10 µm. Red intensities (AMAP1, arbitrary units) and green intensities (PRKD2, arbitrary units) at the sites of gelatin degradation were measured and are shown as a scatter plot (D). (E–H) MDA-MB-231 cells were transfected with PRKD2 siRNA or a dsRNA with a nontargeting, irrelevant sequence (Irr), together with pcDNA3.1 HisC-resPRKD2 (rescue PRKD2) or control pcDNA3 empty vector (−), as indicated (E and F). Hs578T and MDA-MB-435s cells were transfected with PRKD2 siRNA or a dsRNA with an irrelevant sequence (Irr), as indicated (G and H). Total lysates were subjected to blotting with the indicated antibodies (E and G). Activities of cell adhesion to collagen (Adhesion) and Matrigel chemoinvasion (Invasion) are shown (F and H). In F and H, data are presented as percentages calculated by normalizing the values obtained for the control cells as 100%. 4,213 ± 516 (∼4.21%), 2,562 ± 309 (∼2.56%), and 1,790 ± 271 (∼1.79%) control MDA-MB-231, Hs578T, and MDA-MB-435s cells, respectively, were calculated to have transmigrated per 6.4-mm-diam Matrigel-coated Boyden chamber filter under these conditions. Data are shown as mean ± SEM of triplicate experiments (error bars).

    Journal: The Journal of Cell Biology

    Article Title: Rab5c promotes AMAP1-PRKD2 complex formation to enhance ?1 integrin recycling in EGF-induced cancer invasion

    doi: 10.1083/jcb.201201065

    Figure Lengend Snippet: Association of PRKD2 with AMAP1 in highly invasive breast cancer cells. (A) Lysates from different breast cancer cells and HMECs were probed with the indicated antibodies. (B) Lysates from the indicated cells were immunoprecipitated with an anti-AMAP1 antibody or an anti-AMAP2 antibody, then subjected to blotting with the indicated antibodies. Preimmune serum of the anti-AMAP1 antibody was used as a control (Pre-imm). WCL, whole cell lysate (5 µg). (C and D) MDA-MB-231 cells, expressing mVenus-PKD2 and cultured on Alexa Fluor–labeled gelatin film, were fixed and stained with an anti-AMAP1 antibody. Original colors were: PRKD2, green; AMAP1, red; and sites of gelatin degradation, blue (C). Bar, 10 µm. Red intensities (AMAP1, arbitrary units) and green intensities (PRKD2, arbitrary units) at the sites of gelatin degradation were measured and are shown as a scatter plot (D). (E–H) MDA-MB-231 cells were transfected with PRKD2 siRNA or a dsRNA with a nontargeting, irrelevant sequence (Irr), together with pcDNA3.1 HisC-resPRKD2 (rescue PRKD2) or control pcDNA3 empty vector (−), as indicated (E and F). Hs578T and MDA-MB-435s cells were transfected with PRKD2 siRNA or a dsRNA with an irrelevant sequence (Irr), as indicated (G and H). Total lysates were subjected to blotting with the indicated antibodies (E and G). Activities of cell adhesion to collagen (Adhesion) and Matrigel chemoinvasion (Invasion) are shown (F and H). In F and H, data are presented as percentages calculated by normalizing the values obtained for the control cells as 100%. 4,213 ± 516 (∼4.21%), 2,562 ± 309 (∼2.56%), and 1,790 ± 271 (∼1.79%) control MDA-MB-231, Hs578T, and MDA-MB-435s cells, respectively, were calculated to have transmigrated per 6.4-mm-diam Matrigel-coated Boyden chamber filter under these conditions. Data are shown as mean ± SEM of triplicate experiments (error bars).

    Article Snippet: cDNAs and transfection AMAP1 cDNA ( ) was cloned into pEGFP C1 (Takara Bio Inc.). cDNA for PRKD2, amplified by PCR from the first-strand cDNA of MDA-MB-231 cells, was cloned into pmVenus C1 (see below), pcDNA3.1 HisC (Invitrogen), or pCMV-V5 (see below).

    Techniques: Immunoprecipitation, Multiple Displacement Amplification, Expressing, Cell Culture, Labeling, Staining, Transfection, Sequencing, Plasmid Preparation

    Expression of the CortBP1 protein. (A and B) Distribution of the CortBP1 protein in lysates from rat tissues and cell lines. Lysate (1.0 mg) derived from eight adult rat tissues (A) or RAT1 or PC12 cells (B) was incubated with anti-CortBP1 or preimmune serum. Cellular proteins in the immunocomplexes were subjected to Western blot analysis with anti-CortBP1. (A) Lane 1, heart; lane 2, brain; lane 3, spleen; lane 4, lung; lane 5, liver; lane 6, skeletal muscle; lane 7, kidney; lane 8, testis. (C) Expression of full-length CortBP1 in NIH 3T3 cells. Five hundred micrograms of lysates from NIH 3T3 cells (lane 5), NIH 3T3 cells transfected with pcDNA3.1 (lane 6), or NIH 3T3 cells transfected with pcDNA3.62 (lanes 1, 2, 7, and 8) or 1.0 mg of lysates from PC12 cells (lanes 3 and 9) or rat brain (lanes 4 and 10) was incubated with anti-CortBP1 (lanes 5 to 10) or preimmune serum (lanes 1 to 4). Precipitated proteins were subjected to Western blot analysis with anti-CortBP1. The positions of molecular mass markers are indicated in kilodaltons at the left of each panel. The positions of p180 CortBP1 and the heavy chain of immunoglobulin G are indicated with arrowheads and arrows, respectively. IP, immunoprecipitation; IB, immunoblotting.

    Journal: Molecular and Cellular Biology

    Article Title: Identification of a Novel Cortactin SH3 Domain-Binding Protein and Its Localization to Growth Cones of Cultured Neurons

    doi:

    Figure Lengend Snippet: Expression of the CortBP1 protein. (A and B) Distribution of the CortBP1 protein in lysates from rat tissues and cell lines. Lysate (1.0 mg) derived from eight adult rat tissues (A) or RAT1 or PC12 cells (B) was incubated with anti-CortBP1 or preimmune serum. Cellular proteins in the immunocomplexes were subjected to Western blot analysis with anti-CortBP1. (A) Lane 1, heart; lane 2, brain; lane 3, spleen; lane 4, lung; lane 5, liver; lane 6, skeletal muscle; lane 7, kidney; lane 8, testis. (C) Expression of full-length CortBP1 in NIH 3T3 cells. Five hundred micrograms of lysates from NIH 3T3 cells (lane 5), NIH 3T3 cells transfected with pcDNA3.1 (lane 6), or NIH 3T3 cells transfected with pcDNA3.62 (lanes 1, 2, 7, and 8) or 1.0 mg of lysates from PC12 cells (lanes 3 and 9) or rat brain (lanes 4 and 10) was incubated with anti-CortBP1 (lanes 5 to 10) or preimmune serum (lanes 1 to 4). Precipitated proteins were subjected to Western blot analysis with anti-CortBP1. The positions of molecular mass markers are indicated in kilodaltons at the left of each panel. The positions of p180 CortBP1 and the heavy chain of immunoglobulin G are indicated with arrowheads and arrows, respectively. IP, immunoprecipitation; IB, immunoblotting.

    Article Snippet: To express full-length CortBP1 in mammalian cells, pcDNA3.6 and pcDNA3.2 were generated by subcloning the Not I-digested inserts of phage DNA clones 6.b and 2.b (see Fig. A) into Not I-excised pcDNA3.1(−) (Invitrogen).

    Techniques: Expressing, Derivative Assay, Incubation, Western Blot, Transfection, Immunoprecipitation

    Overexpression of Rab1 protein antagonizes the inhibitory effect of radicicol on VSV-G ts transport. (A) CHO-K1 cells were transfected with pcDNA3.1 plasmids containing Rab1B, Rab3A, Sar1A, or empty vector 24 h before infection with VSV ts as described in Materials and Methods . Infected cells were then pulse-labeled in the absence or presence of radicicol (Rad). Representative autoradiograph is shown. (B) Quantitation based on two independent experiments with triplicate samples. Reported as percentage of increase in endo H-resistant VSV-G ts normalized to mock control to illustrate fold-difference in recovery. (C) Immunoblot analysis of transfected cell lysates demonstrating expression of each of the GTPases relative to endogenous levels. Transient expression did not significantly affect the endogenous level of Hsp90, GDI or alter the total amount of VSV-G ts synthesized.

    Journal: Molecular Biology of the Cell

    Article Title: The Hsp90 Chaperone Complex Regulates GDI-dependent Rab Recycling

    doi: 10.1091/mbc.E05-12-1096

    Figure Lengend Snippet: Overexpression of Rab1 protein antagonizes the inhibitory effect of radicicol on VSV-G ts transport. (A) CHO-K1 cells were transfected with pcDNA3.1 plasmids containing Rab1B, Rab3A, Sar1A, or empty vector 24 h before infection with VSV ts as described in Materials and Methods . Infected cells were then pulse-labeled in the absence or presence of radicicol (Rad). Representative autoradiograph is shown. (B) Quantitation based on two independent experiments with triplicate samples. Reported as percentage of increase in endo H-resistant VSV-G ts normalized to mock control to illustrate fold-difference in recovery. (C) Immunoblot analysis of transfected cell lysates demonstrating expression of each of the GTPases relative to endogenous levels. Transient expression did not significantly affect the endogenous level of Hsp90, GDI or alter the total amount of VSV-G ts synthesized.

    Article Snippet: All expression plasmids were generated using a pc3.1 vector (Invitrogen).

    Techniques: Over Expression, Transfection, Plasmid Preparation, Infection, Labeling, Autoradiography, Quantitation Assay, Expressing, Synthesized

    Analysis of the particle association of the Env-Bet fusion protein. Following transfection with different proviral expression constructs, 293T cells were metabolically labeled, and the appearance of HFV Env proteins in the supernatant and associated with viral particles was analyzed. (A) RIPA of viral proteins secreted into the supernatant by using an anti-HFV chimpanzee serum. On the left is a longer exposure of lanes 1 and 2. (B) Particle-associated proteins after pelleting through 20% sucrose. Lanes: 1, pcHSRV2 wt; 2, pcHSRV2 EM4; 3, pcHSRV2 EM6; 4, pcHSRV2 EM7; 5, pcHSRV2 EM8; 6, pcHSRV2 EM9; 7, mock, pCDNA3.1+zeo.

    Journal: Journal of Virology

    Article Title: Characterization of a Human Foamy Virus 170-Kilodalton Env-Bet Fusion Protein Generated by Alternative Splicing

    doi:

    Figure Lengend Snippet: Analysis of the particle association of the Env-Bet fusion protein. Following transfection with different proviral expression constructs, 293T cells were metabolically labeled, and the appearance of HFV Env proteins in the supernatant and associated with viral particles was analyzed. (A) RIPA of viral proteins secreted into the supernatant by using an anti-HFV chimpanzee serum. On the left is a longer exposure of lanes 1 and 2. (B) Particle-associated proteins after pelleting through 20% sucrose. Lanes: 1, pcHSRV2 wt; 2, pcHSRV2 EM4; 3, pcHSRV2 EM6; 4, pcHSRV2 EM7; 5, pcHSRV2 EM8; 6, pcHSRV2 EM9; 7, mock, pCDNA3.1+zeo.

    Article Snippet: The expression construct pcHFVenv/bel1-3, containing the env , bel1 , bel2 , and bel3 ORFs, was generated by inserting a fragment of pHSRV2 ( ) from the translation start of the env ORF (nucleotide [nt] 5719 relative to the genomic transcription start) to the Ssp I site (nt 10445) ∼35 bp downstream of the bel2 stop codon into pCDNA3.1+zeo (Invitrogen).

    Techniques: Transfection, Expressing, Construct, Metabolic Labelling, Labeling

    Identification of splice variants of canine mda- 7. Canine mda- 7 splice variants were amplified by nested PCR and cloned into the pCDNA3.1+/Hygro plasmid vector. Recombinant clones were digested with Hind III and Xho I restriction enzymes to release the

    Journal: Gene

    Article Title: Characterization of the canine mda-7 gene, transcripts and expression patterns

    doi: 10.1016/j.gene.2014.05.054

    Figure Lengend Snippet: Identification of splice variants of canine mda- 7. Canine mda- 7 splice variants were amplified by nested PCR and cloned into the pCDNA3.1+/Hygro plasmid vector. Recombinant clones were digested with Hind III and Xho I restriction enzymes to release the

    Article Snippet: Different splice variants were amplified using nested PCR and cloned into pGEMT easy and pCDNA3.1+/Hygro (Invitrogen, Inc.) vectors.

    Techniques: Multiple Displacement Amplification, Amplification, Nested PCR, Clone Assay, Plasmid Preparation, Recombinant

    Effects of NSPc1 on HeLa cell cycle progression, cell proliferation and cell colony formation. ( a ) Establishment of the NSPc1 stably over-expressed and NSPc1 knocked-down HeLa cell pools. Up: changes in mRNA level of HeLa stably transfected cells pool confirmed by semi-quantitative RT–PCR. PCR products generated by primer sets for NSPc1 were normalized by glyceraldehyde-3-phosphate dehydrogenase (GAPDH); Down: changes in protein expression confirmed by Western blots (normalized by β-actin protein level). ( b and c ) Flow cytometric analysis of HeLa stable cell pools. ( d and e ) MTT assay of HeLa stable cell pools. ( f and g ) Colony formation assay. Culture dishes fixed and stained with 0.1% crystal violet at the end of the experiment. The numbers of total colonies and colonies greater than 2 mm in diameter were counted and the means and SDs of three independent experiments were shown. Abbreviations: V5, pcDNA3.1-V5 cell pool; NSPc1, pcDNA3.1-V5+NSPc1 cell pool; IMG800, IMG800 cell pool; RNAi1, NSPc1-RNAi1 cell pool; RNAi2, NSPc1-RNAi2 cell pool. Note: a representative experiment out of three performed is shown in (b–f).

    Journal: Nucleic Acids Research

    Article Title: NSPc1 is a cell growth regulator that acts as a transcriptional repressor of p21Waf1/Cip1 via the RARE element

    doi: 10.1093/nar/gkl834

    Figure Lengend Snippet: Effects of NSPc1 on HeLa cell cycle progression, cell proliferation and cell colony formation. ( a ) Establishment of the NSPc1 stably over-expressed and NSPc1 knocked-down HeLa cell pools. Up: changes in mRNA level of HeLa stably transfected cells pool confirmed by semi-quantitative RT–PCR. PCR products generated by primer sets for NSPc1 were normalized by glyceraldehyde-3-phosphate dehydrogenase (GAPDH); Down: changes in protein expression confirmed by Western blots (normalized by β-actin protein level). ( b and c ) Flow cytometric analysis of HeLa stable cell pools. ( d and e ) MTT assay of HeLa stable cell pools. ( f and g ) Colony formation assay. Culture dishes fixed and stained with 0.1% crystal violet at the end of the experiment. The numbers of total colonies and colonies greater than 2 mm in diameter were counted and the means and SDs of three independent experiments were shown. Abbreviations: V5, pcDNA3.1-V5 cell pool; NSPc1, pcDNA3.1-V5+NSPc1 cell pool; IMG800, IMG800 cell pool; RNAi1, NSPc1-RNAi1 cell pool; RNAi2, NSPc1-RNAi2 cell pool. Note: a representative experiment out of three performed is shown in (b–f).

    Article Snippet: Establishment of NSPc1 stably integrated cell pools and NSPc1 knocked down cell pools To establish NSPc1 stably integrated HeLa cell pools, the coding sequence of NSPc1 was inserted into pcDNA3.1-V5 (Invitrogen, USA) for transfection into HeLa cells.

    Techniques: Stable Transfection, Transfection, Quantitative RT-PCR, Polymerase Chain Reaction, Generated, Expressing, Western Blot, Flow Cytometry, MTT Assay, Colony Assay, Staining

    Flow cytometric analysis of HeLa and SH-SY5Y cells transiently transfected with pcDNA3.1-v5+NSPc1 or control vector. A representative experiment out of at least three performed is shown both in cell phase distribution and bar diagrams. ( a–c ) HeLa cell, ( d–f ) SH-SY5Y cell.

    Journal: Nucleic Acids Research

    Article Title: NSPc1 is a cell growth regulator that acts as a transcriptional repressor of p21Waf1/Cip1 via the RARE element

    doi: 10.1093/nar/gkl834

    Figure Lengend Snippet: Flow cytometric analysis of HeLa and SH-SY5Y cells transiently transfected with pcDNA3.1-v5+NSPc1 or control vector. A representative experiment out of at least three performed is shown both in cell phase distribution and bar diagrams. ( a–c ) HeLa cell, ( d–f ) SH-SY5Y cell.

    Article Snippet: Establishment of NSPc1 stably integrated cell pools and NSPc1 knocked down cell pools To establish NSPc1 stably integrated HeLa cell pools, the coding sequence of NSPc1 was inserted into pcDNA3.1-V5 (Invitrogen, USA) for transfection into HeLa cells.

    Techniques: Flow Cytometry, Transfection, Plasmid Preparation

    FUS promotes miR-200c-mediated degradation of ZEB1 (A) Bar graphs show the amount of immunoprecipitated miR-200c (n=5). HEK293 cells are transfected with myc-FUS, myc-R495X, myc-SOD1, or control pcDNA3.1, along with pCMV-miR-200c, prior to the immunoprecipitation by anti-myc antibodies. (B) Bar graph represents enrichment of mature miR-200c in RIP experiments using a FUS antibody in human WT HAP1 cells versus FUS KO HAP1 cells (n=3), indicating the endogenous interaction between FUS and miR-200c. (C) Bar graphs show the amount of immunoprecipitated mRNA of ZEB1 (left) and FZD6 (right). HEK293 cells are transfected with pcDNA3.1, myc-FUS or myc-R495X, along with pCMV-miR-200c, prior to immunoprecipitation with anti-myc antibodies. (D) Bar graphs show the amount of immunoprecipitated mRNA of ZEB1 in HEK293 cells transfected with FUS-V5 or LacZ-V5 together with a specific miR-200c antagomir or a control antagomir, prior to immunoprecipitation with anti-V5 antibodies. (E) (top) A model of the experimental design to test the role of FUS or miR-200c binding to ZEB1 3′ UTR in its silencing. Bar graphs compare the normalized luciferase activity of the pmirGLO reporters with WT or mutated ZEB1 3′ UTRs in the presence of recombinant pCMV-miR-505 or pCMV-miR-200c, plotted as a percentage relative to the control (n=3). Normalization was performed by dividing the firefly activity by the Renilla .

    Journal: Molecular cell

    Article Title: FUS Regulates Activity of MicroRNA-mediated Gene Silencing

    doi: 10.1016/j.molcel.2018.02.001

    Figure Lengend Snippet: FUS promotes miR-200c-mediated degradation of ZEB1 (A) Bar graphs show the amount of immunoprecipitated miR-200c (n=5). HEK293 cells are transfected with myc-FUS, myc-R495X, myc-SOD1, or control pcDNA3.1, along with pCMV-miR-200c, prior to the immunoprecipitation by anti-myc antibodies. (B) Bar graph represents enrichment of mature miR-200c in RIP experiments using a FUS antibody in human WT HAP1 cells versus FUS KO HAP1 cells (n=3), indicating the endogenous interaction between FUS and miR-200c. (C) Bar graphs show the amount of immunoprecipitated mRNA of ZEB1 (left) and FZD6 (right). HEK293 cells are transfected with pcDNA3.1, myc-FUS or myc-R495X, along with pCMV-miR-200c, prior to immunoprecipitation with anti-myc antibodies. (D) Bar graphs show the amount of immunoprecipitated mRNA of ZEB1 in HEK293 cells transfected with FUS-V5 or LacZ-V5 together with a specific miR-200c antagomir or a control antagomir, prior to immunoprecipitation with anti-V5 antibodies. (E) (top) A model of the experimental design to test the role of FUS or miR-200c binding to ZEB1 3′ UTR in its silencing. Bar graphs compare the normalized luciferase activity of the pmirGLO reporters with WT or mutated ZEB1 3′ UTRs in the presence of recombinant pCMV-miR-505 or pCMV-miR-200c, plotted as a percentage relative to the control (n=3). Normalization was performed by dividing the firefly activity by the Renilla .

    Article Snippet: To perform RNA immunoprecipitation with proteins, following 24–48 hour transfection of HEK293 cells with myc-FUS, myc-R495X, myc-SOD1, or empty pcDNA3.1 (Invitrogen), and pCMV-miR-200c/miR-505 (Life Technologies) via Lipofectamine 2000, cells were lysed with polysome lysis buffer.

    Techniques: Immunoprecipitation, Transfection, Binding Assay, Luciferase, Activity Assay, Recombinant

    Constitutively expressed MEG3 suppresses xenograft tumor growth in nude mice. PDFS cells were transfected with pcDNA3.1-puro vector, pCMV-MEG3-puro or pCMV-MEG3del5-puro constructs. Puromycin resistant clones were isolated. MEG3 (A) or MEG3del5 (B) expression

    Journal: Molecular and cellular endocrinology

    Article Title: Tumor Suppression by MEG3 lncRNA in a Human Pituitary Tumor Derived Cell Line

    doi: 10.1016/j.mce.2015.08.018

    Figure Lengend Snippet: Constitutively expressed MEG3 suppresses xenograft tumor growth in nude mice. PDFS cells were transfected with pcDNA3.1-puro vector, pCMV-MEG3-puro or pCMV-MEG3del5-puro constructs. Puromycin resistant clones were isolated. MEG3 (A) or MEG3del5 (B) expression

    Article Snippet: Plasmid pcDNA3.1 was obtained from Life Technologies (Grand Island, NY). pSuper.Retro.puro and pSuper.p53 were obtained from OligoEngine (Seattle, WA).

    Techniques: Mouse Assay, Transfection, Plasmid Preparation, Construct, Clone Assay, Isolation, Expressing

    Effect of baicalein on the ERK pathway. (A) The protein levels of MEK1, p-MEK1, ERK1/2 and p-ERK1/2. (B) Phosphorylation densities of MEK1 and ERK1/2 were digitally scanned. (C) Transwell assays were performed to evaluate the anti-metastatic effects of baicalein on two groups of cells transfected with the indicated plasmids (transfected with the empty vector pcDNA3.1 (+) or with pcDNA3.1 (+)-MEK1, ‘pcDNA3.1(+)’ means the group transfected with an empty vector pcDNA3.1(+), ‘pcDNA3.1(+)-MEK1’ means the group transfected with a pcDNA3.1(+)-MEK1). (D) The percent invasion rate was expressed as a percentage of the control (the control group of cells transfected with an empty vector pcDNA3.1 (+)). (E) The inhibition rates of baicalein on two groups of cells. Values represent the means ± SD of three independent experiments performed in triplicate. * p

    Journal: PLoS ONE

    Article Title: Baicalein Inhibits the Invasion and Metastatic Capabilities of Hepatocellular Carcinoma Cells via Down-Regulation of the ERK Pathway

    doi: 10.1371/journal.pone.0072927

    Figure Lengend Snippet: Effect of baicalein on the ERK pathway. (A) The protein levels of MEK1, p-MEK1, ERK1/2 and p-ERK1/2. (B) Phosphorylation densities of MEK1 and ERK1/2 were digitally scanned. (C) Transwell assays were performed to evaluate the anti-metastatic effects of baicalein on two groups of cells transfected with the indicated plasmids (transfected with the empty vector pcDNA3.1 (+) or with pcDNA3.1 (+)-MEK1, ‘pcDNA3.1(+)’ means the group transfected with an empty vector pcDNA3.1(+), ‘pcDNA3.1(+)-MEK1’ means the group transfected with a pcDNA3.1(+)-MEK1). (D) The percent invasion rate was expressed as a percentage of the control (the control group of cells transfected with an empty vector pcDNA3.1 (+)). (E) The inhibition rates of baicalein on two groups of cells. Values represent the means ± SD of three independent experiments performed in triplicate. * p

    Article Snippet: Briefly, we made the full-length pcDNA3.1 (Invitrogen) MEK1 vector by cloning the full-length PCR product of MEK1 with KOD® DNA polymerase (Toyobo, Osaka, Japan).

    Techniques: Transfection, Plasmid Preparation, Inhibition

    Evaluation of in vitro DNA binding of Hey1 to myogenic promoter elements. A , TNT lysates programmed with either pcDNA3.1-V5/HisA empty vector or pcDNA3.1-TOPO-Hey1-V5 were incubated with a 22-nucleotide 32 P-labeled probe containing the Hey1 consensus target E-box (CACGTG). Anti-V5 antibodies and cold competitor probes (50× excess relative to labeled probe) were added as indicated before resolution of complexes by non-denaturing SDS-PAGE.; Δ HCE , mutant Hey1-consensus E-box; E1 , E2 , E-boxes within the myogenin proximal promoter; N1 , N-box ∼400 bp upstream of myogenin start site; 2C , high affinity MyoD E-box within the Mef2C proximal promoter. B , 10T1/2 cells were transfected with 25 ng of pRL-tk-Renilla, 25 ng of G133-luciferase or G133-mutE1-luciferase, 25 ng of pEMSV-MyoD, and 25–100 ng of pcDNA3.1-Hey1-V5. Firefly luciferase values were normalized to Renilla luciferase and plotted as the averages of three replicate samples ±S.D.

    Journal: The Journal of Biological Chemistry

    Article Title: The Notch Effector Hey1 Associates with Myogenic Target Genes to Repress Myogenesis *

    doi: 10.1074/jbc.M109.046441

    Figure Lengend Snippet: Evaluation of in vitro DNA binding of Hey1 to myogenic promoter elements. A , TNT lysates programmed with either pcDNA3.1-V5/HisA empty vector or pcDNA3.1-TOPO-Hey1-V5 were incubated with a 22-nucleotide 32 P-labeled probe containing the Hey1 consensus target E-box (CACGTG). Anti-V5 antibodies and cold competitor probes (50× excess relative to labeled probe) were added as indicated before resolution of complexes by non-denaturing SDS-PAGE.; Δ HCE , mutant Hey1-consensus E-box; E1 , E2 , E-boxes within the myogenin proximal promoter; N1 , N-box ∼400 bp upstream of myogenin start site; 2C , high affinity MyoD E-box within the Mef2C proximal promoter. B , 10T1/2 cells were transfected with 25 ng of pRL-tk-Renilla, 25 ng of G133-luciferase or G133-mutE1-luciferase, 25 ng of pEMSV-MyoD, and 25–100 ng of pcDNA3.1-Hey1-V5. Firefly luciferase values were normalized to Renilla luciferase and plotted as the averages of three replicate samples ±S.D.

    Article Snippet: Mef2C-luciferase was generated by PCR amplification of the Mef2C proximal promoter (−158 to +106) from genomic DNA and insertion into the KpnI/BglII sites of pGL3-basic (Promega, Madison, WI). pcDNA3.1-TOPO- Hey1-V5 (C-terminal V5 epitope) was generated by inserting the full-length Hey1 cDNA (provided by Eric Olson) into the TOPO recognition site of pcDNA3.1D/V5-His-TOPO (Invitrogen). pcDNA3.1-Hey1-V5 (C-terminal V5 epitope) was generated by PCR subcloning the full-length Hey1 cDNA into the BamHI/EcoR1 sites of pcDNA3.1-V5/HisA (Invitrogen). pcDNA-Myc-Hey1 (N-terminal Myc epitope) has been described previously ( ).

    Techniques: In Vitro, Binding Assay, Plasmid Preparation, Incubation, Labeling, SDS Page, Mutagenesis, Transfection, Luciferase

    COSMC modulates protein degradation of VEGFR2. (A) COSMC overexpression delays degradation of VEGFR2. HUVECs were treated with cycloheximide (10 µg/ml) to block protein synthesis and 20 ng/ml of VEGF to trigger internalization and degradation of VEGFR2 for indicated time points. Upper panel shows representative Western blots. Lower panel shows signals on Western blots quantified by ImageQuant5.1 for HUVECs transfected with pcDNA3.1 control plasmid (dashed line) and UVECs transfected with COSMC/pcDNA3.1 (solid line). (B) COSMC knockdown facilitates degradation of VEGFR2. VEGFR2 degradation in HUVECs transfected with control siRNA (dashed line) or COSMC siRNA (solid line) was shown. Representative data from two independent experiments are presented.

    Journal: PLoS ONE

    Article Title: COSMC Is Overexpressed in Proliferating Infantile Hemangioma and Enhances Endothelial Cell Growth via VEGFR2

    doi: 10.1371/journal.pone.0056211

    Figure Lengend Snippet: COSMC modulates protein degradation of VEGFR2. (A) COSMC overexpression delays degradation of VEGFR2. HUVECs were treated with cycloheximide (10 µg/ml) to block protein synthesis and 20 ng/ml of VEGF to trigger internalization and degradation of VEGFR2 for indicated time points. Upper panel shows representative Western blots. Lower panel shows signals on Western blots quantified by ImageQuant5.1 for HUVECs transfected with pcDNA3.1 control plasmid (dashed line) and UVECs transfected with COSMC/pcDNA3.1 (solid line). (B) COSMC knockdown facilitates degradation of VEGFR2. VEGFR2 degradation in HUVECs transfected with control siRNA (dashed line) or COSMC siRNA (solid line) was shown. Representative data from two independent experiments are presented.

    Article Snippet: COSMC/pcDNA3.1 (a gift from Dr. Richard D. Cummings at the Emory University School of Medicine, Atlanta, USA) and control pcDNA3.1 (Mock) (Invitrogen, Carlsbad, CA) were transfected into HUVECs (3-5 passages) using an Amaxa Nucleofector™ (Lonza, Walkersville, MD) and the HUVEC Nucleofector kit (Lonza, Walkersville, MD) according to the manufacturer's instructions.

    Techniques: Over Expression, Blocking Assay, Western Blot, Transfection, Plasmid Preparation

    COSMC overexpression enhances cell growth in HUVECs. (A) Cell growth of HUVECs transfected with pcDNA3.1 control plasmid (open bars) or COSMC/pcDNA3.1 (closed bars) analyzed by trypan blue exclusion assays. (B) Cell growth of HUVECs transfected with control siRNA (open bars) or COSMC siRNA (closed bars) analyzed by trypan blue exclusion assays. (C) Cell growth of EA.hy926 cells overexpressing COSMC. (D) Cell growth of EA.hy926 cells with COSMC knockdown. Results are presented as means ± SD from three independent experiments. * P

    Journal: PLoS ONE

    Article Title: COSMC Is Overexpressed in Proliferating Infantile Hemangioma and Enhances Endothelial Cell Growth via VEGFR2

    doi: 10.1371/journal.pone.0056211

    Figure Lengend Snippet: COSMC overexpression enhances cell growth in HUVECs. (A) Cell growth of HUVECs transfected with pcDNA3.1 control plasmid (open bars) or COSMC/pcDNA3.1 (closed bars) analyzed by trypan blue exclusion assays. (B) Cell growth of HUVECs transfected with control siRNA (open bars) or COSMC siRNA (closed bars) analyzed by trypan blue exclusion assays. (C) Cell growth of EA.hy926 cells overexpressing COSMC. (D) Cell growth of EA.hy926 cells with COSMC knockdown. Results are presented as means ± SD from three independent experiments. * P

    Article Snippet: COSMC/pcDNA3.1 (a gift from Dr. Richard D. Cummings at the Emory University School of Medicine, Atlanta, USA) and control pcDNA3.1 (Mock) (Invitrogen, Carlsbad, CA) were transfected into HUVECs (3-5 passages) using an Amaxa Nucleofector™ (Lonza, Walkersville, MD) and the HUVEC Nucleofector kit (Lonza, Walkersville, MD) according to the manufacturer's instructions.

    Techniques: Over Expression, Transfection, Plasmid Preparation