pcdna flag  (Thermo Fisher)


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    Structured Review

    Thermo Fisher pcdna flag
    Expression of ZIKV NS5 induces degradation of STAT2 A549 cells were transfected with control plasmid <t>pcDNA</t> 3.1 or plasmids encoding <t>FLAG‐tagged</t> ZIKV proteins. At 48 h, post‐transfection cells were treated with IFN‐α (100 U/ml) for 2 h and then processed for indirect immunofluorescence. Images were acquired on a spinning disk confocal microscope with a 40× objective. NS5‐positive cells are indicated by dashed white circles. Representative panels are shown. The experiment was repeated three times.
    Pcdna Flag, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Zika virus inhibits type‐I interferon production and downstream signaling"

    Article Title: Zika virus inhibits type‐I interferon production and downstream signaling

    Journal: EMBO Reports

    doi: 10.15252/embr.201642627

    Expression of ZIKV NS5 induces degradation of STAT2 A549 cells were transfected with control plasmid pcDNA 3.1 or plasmids encoding FLAG‐tagged ZIKV proteins. At 48 h, post‐transfection cells were treated with IFN‐α (100 U/ml) for 2 h and then processed for indirect immunofluorescence. Images were acquired on a spinning disk confocal microscope with a 40× objective. NS5‐positive cells are indicated by dashed white circles. Representative panels are shown. The experiment was repeated three times.
    Figure Legend Snippet: Expression of ZIKV NS5 induces degradation of STAT2 A549 cells were transfected with control plasmid pcDNA 3.1 or plasmids encoding FLAG‐tagged ZIKV proteins. At 48 h, post‐transfection cells were treated with IFN‐α (100 U/ml) for 2 h and then processed for indirect immunofluorescence. Images were acquired on a spinning disk confocal microscope with a 40× objective. NS5‐positive cells are indicated by dashed white circles. Representative panels are shown. The experiment was repeated three times.

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

    Related Articles

    Sequencing:

    Article Title: An Adenovirus Inhibitor of Tumor Necrosis Factor Alpha-Induced Apoptosis Complexes with Dynein and a Small GTPase
    Article Snippet: .. Construction of pcDNA-T7 and pcDNA-FLAG from pcDNA-3 (Invitrogen) has been previously described ( ). pcDNA-T7-GIP-1 was made by excising the GIP-1 cDNA from the pGAD vector at the Bam HI and Xho I sites and inserting it in pcDNA-T7 linearized with Bam HI and Xhol I. pcDNA-FLAG-FIP-1 was made by excising the FIP-1 cDNA from pBMT-FIP-1 at the Eco RI and Xho I restriction sites and cloning it into the pcDNA-FLAG plasmid with FLAG expressed at the 5′ end of the FIP-1 coding sequence. .. For the glutathione S -transferase (GST) assay, the pGEX-FIP-1 construct, previously described ( ) was used. pCITE-GIP-1 was constructed by inserting the GIP-1 clone into the pCITE plasmid (Novagen) at the Bam HI and Xho I sites.

    Clone Assay:

    Article Title: An Adenovirus Inhibitor of Tumor Necrosis Factor Alpha-Induced Apoptosis Complexes with Dynein and a Small GTPase
    Article Snippet: .. Construction of pcDNA-T7 and pcDNA-FLAG from pcDNA-3 (Invitrogen) has been previously described ( ). pcDNA-T7-GIP-1 was made by excising the GIP-1 cDNA from the pGAD vector at the Bam HI and Xho I sites and inserting it in pcDNA-T7 linearized with Bam HI and Xhol I. pcDNA-FLAG-FIP-1 was made by excising the FIP-1 cDNA from pBMT-FIP-1 at the Eco RI and Xho I restriction sites and cloning it into the pcDNA-FLAG plasmid with FLAG expressed at the 5′ end of the FIP-1 coding sequence. .. For the glutathione S -transferase (GST) assay, the pGEX-FIP-1 construct, previously described ( ) was used. pCITE-GIP-1 was constructed by inserting the GIP-1 clone into the pCITE plasmid (Novagen) at the Bam HI and Xho I sites.

    Transfection:

    Article Title: Zika virus inhibits type‐I interferon production and downstream signaling
    Article Snippet: .. A549 cells were transfected with pCDNA NS5‐FLAG, NS5 mutants, or pCDNA‐FLAG with Lipofectamine 2000 (Life Technologies) for indicated time periods with treatments described. .. The cells were pelleted and resuspended in IP buffer (137 mM NaCl, 50 mM Tris pH 7.5, 1% NP‐40, 1 mM NaF, 1 mM DTT, and protease inhibitor cocktail (Roche)).

    Mutagenesis:

    Article Title: The Fungal Metabolite Brefeldin A Inhibits Dvl2-Plk1-Dependent Primary Cilium Disassembly
    Article Snippet: .. Plasmid construction A Bam HI-Eco RI fragment of mouse Dvl2 S594E, T595E, and S597E mutant (3E) was sub-cloned into pcDNA-Flag (Invitrogen, USA). .. The Bam HI-Eco RI fragment of mouse Dvl23E was generated by polymerase chain reaction (PCR) using pcDNA-Flag-Dvl2 wild-type (WT) as a template.

    Article Title: Phosphorylation of human enhancer filamentation 1 (HEF1) stimulates interaction with Polo-like kinase 1 leading to HEF1 localization to focal adhesions
    Article Snippet: .. Plasmid construction and mutagenesis The human, WT form of HEF1 , or various mutant forms (each a truncation mutant) and an alanine substitution mutant were subcloned into the KpnI-NotI site of pcDNA-FLAG (Invitrogen), the EcoRI-NotI site of pGEX-4T-2 (Amersham Biosciences), or the SalI site of pHR ′-CMV-SV-puro (a gift from Chou-Zen Giam, Uniformed Services University of the Health Sciences, Bethesda, MD). .. Each full-length and truncation fragment was generated by PCR, using the pCMV-FLAG-HEF1 WT (a gift from Joel Raingeaud, INSERM, France) as a template.

    Plasmid Preparation:

    Article Title: TRPC5 channel instability induced by depalmitoylation protects striatal neurons against oxidative stress in Huntington's disease.
    Article Snippet: .. Protein S-palmitoylation, the covalent lipid modification of the side chain of Cys residues with the 16‑carbon fatty acid palmitate, is the most common acylation, and it enhances the membrane stability of ion channels. .. Protein S-palmitoylation, the covalent lipid modification of the side chain of Cys residues with the 16‑carbon fatty acid palmitate, is the most common acylation, and it enhances the membrane stability of ion channels.

    Article Title: An Adenovirus Inhibitor of Tumor Necrosis Factor Alpha-Induced Apoptosis Complexes with Dynein and a Small GTPase
    Article Snippet: .. Construction of pcDNA-T7 and pcDNA-FLAG from pcDNA-3 (Invitrogen) has been previously described ( ). pcDNA-T7-GIP-1 was made by excising the GIP-1 cDNA from the pGAD vector at the Bam HI and Xho I sites and inserting it in pcDNA-T7 linearized with Bam HI and Xhol I. pcDNA-FLAG-FIP-1 was made by excising the FIP-1 cDNA from pBMT-FIP-1 at the Eco RI and Xho I restriction sites and cloning it into the pcDNA-FLAG plasmid with FLAG expressed at the 5′ end of the FIP-1 coding sequence. .. For the glutathione S -transferase (GST) assay, the pGEX-FIP-1 construct, previously described ( ) was used. pCITE-GIP-1 was constructed by inserting the GIP-1 clone into the pCITE plasmid (Novagen) at the Bam HI and Xho I sites.

    Article Title: The Fungal Metabolite Brefeldin A Inhibits Dvl2-Plk1-Dependent Primary Cilium Disassembly
    Article Snippet: .. Plasmid construction A Bam HI-Eco RI fragment of mouse Dvl2 S594E, T595E, and S597E mutant (3E) was sub-cloned into pcDNA-Flag (Invitrogen, USA). .. The Bam HI-Eco RI fragment of mouse Dvl23E was generated by polymerase chain reaction (PCR) using pcDNA-Flag-Dvl2 wild-type (WT) as a template.

    Article Title: Phosphorylation of human enhancer filamentation 1 (HEF1) stimulates interaction with Polo-like kinase 1 leading to HEF1 localization to focal adhesions
    Article Snippet: .. Plasmid construction and mutagenesis The human, WT form of HEF1 , or various mutant forms (each a truncation mutant) and an alanine substitution mutant were subcloned into the KpnI-NotI site of pcDNA-FLAG (Invitrogen), the EcoRI-NotI site of pGEX-4T-2 (Amersham Biosciences), or the SalI site of pHR ′-CMV-SV-puro (a gift from Chou-Zen Giam, Uniformed Services University of the Health Sciences, Bethesda, MD). .. Each full-length and truncation fragment was generated by PCR, using the pCMV-FLAG-HEF1 WT (a gift from Joel Raingeaud, INSERM, France) as a template.

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    Thermo Fisher pcdna3 flag ha dcaf11
    CUL4B interacted with DDB1, RXB1 and <t>DCAF11</t> to form a complex in vivo and in vitro . ( A ) Flag-tagged CUL4B interacted with RBX1 and DDB1 in vivo . The <t>pCDNA3-CUL4B-Flag</t> or pCDNA3-Flag empty vector was transformed into hFOB1.19, U2OS or Saos-2 cells. After 48 h incubation, Flag-tagged proteins were immunoprecipitated with anti-Flag agarose. The pull-down products were analysed by immunoblotting with anti-Flag, anti-RBX1, or anti-DDB1 antibodies. ( B ) Interactions between CUL4B/DDB1 and CUL4B/RBX1 in yeast. The pGBKT7-CUL4B plasmid was co-transformed with pGADT7-DDB1 or pGADT7-RBX1 into the yeast strain AH109. Growth of the transformed yeast was assayed on media lacking Trp and Leu (top panel) or lacking Trp, Leu, and His (bottom panel). Columns in each panel represent serial 10-fold dilutions. ( C ) Flag-tagged CUL4B associated with DCAF11 in vivo . The pCDNA3-CUL4B-Flag plasmid was transformed into hFOB1.19 (1), U2OS (2) or Saos-2 (3) cells. After 48 h incubation, Flag-tagged proteins were immunoprecipitated with either protein A agarose or anti-Flag-agarose. The cell lysate and pull-down products were analysed by immunoblotting with anti-CUL4B, anti-DCAF1, anti-DCAF4, anti-DCAF8, anti-DCAF11 or anti-DCAF15 antibodies. ( D ) DCAF11 was overexpressed in human osteosarcoma cell lines. Cell lysates were applied to WB analyses with specific antibodies against CUL4B, DDB1, RBX1, DCAF11, DCAF4, or β-Actin.
    Pcdna3 Flag Ha Dcaf11, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermo Fisher pcdna3 1 flag usp25fl c178a
    The USP25 dimer stabilizes endogenous tankyrase in HEK293T cells. a <t>Flag-USP25FL,</t> Flag-USP25FL ΔIL, Flag-USP25FL <t>C178A,</t> Flag-USP25FL ΔIL C178A, Flag-USP25NCD, and Flag-USP25NCD ΔIL were transfected in HEK293T cells and the levels of endogenous tankyrases1/2 were analyzed by western blot (WB). GFP was transfected as a control. Plot of the quantification of tankyrase1/2 levels, corrected with tubulin and relative to GFP. Data values are mean ± s.d. and n ≥ 3 technical replicates. Significance was measured by a two-tailed unpaired t test for all lanes relative to GFP and between USP25FL and USP25FL ΔIL. * P
    Pcdna3 1 Flag Usp25fl C178a, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Thermo Fisher pcdna3 1 flag sbd
    Effect of mutations in the N-terminal region of the <t>SBD</t> on binding with PHD3. HEK293T cells were transfected with the expression plasmids for the proteins indicated at the top of each panel. The cells were lysed and the cell lysates were subjected to <t>FLAG-immunoprecipiation</t> (IP). Immunoprecipitates and lysates were then analyzed by western blotting using the indicated antibodies. ( a ) Both [S1-(P21H/A26P/Q62A)] NT [S2] CT and [S1-6Mut] NT [S2] CT chimeras only partially regained binding to PHD3, compared to binding of wild type Siah2 SBD to PHD3. ( b ) Only the chimera with the P21H/A26P mutations regained partial binding with PHD3. In contrast, mutation of Q62A did not increase PHD3 binding. FLAG-SBD Siah in the IP was masked by the IgG light chain.
    Pcdna3 1 Flag Sbd, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Thermo Fisher pcdna3 1 slug flag
    LSD1 acts cooperatively with Slug to inactivate ESR1 promoters by demethylating H3K4me2. ( a , b ) Western blot ( a ) and RT–PCR ( b ) analyze effects of LSD1 and Slug on ERα expression via Slug overexpression and LSD1 knockdown in MCF-7 cells. ( c , d ) Western blot ( c ) and RT–PCR ( d ) analysis of effects of LSD1 and Slug on ERα expression via Slug and LSD1 knockdown in MDA-MB-231 cells. ( e ) MDA-MB-231 cells expressing pHAGE-CMV-Flag-HA-LSD1 and <t>pcDNA3.1-Slug-Flag</t> or pcDNA3.1-ΔN-Slug-Flag was analyzed by IP. Cell lysates were immunoprecipitated with anti-HA antibody. Immunocomplexes were then immunoblotted using antibodies against Flag and HA. ( f ) A dual-luciferase reporter assay is used to investigate ESR1 promoter 2 activity in MDA-MB-231 cells by overexpressing wild-type or mutant Slug. ( g ) ChIP-qPCR analysis of LSD1 recruitment on ESR1 promoter in MDA-MB-231shSlug cells and their control cells. ( h ) ChIP-qPCR analysis of H3K4me2 recruitment on ESR1 promoter in MDA-MB-231shLSD1 cells and their control cells. All the ChIP-qPCR results represent % of input chromatin. ( i ) A dual-luciferase reporter assay is used to investigate ESR1 promoter activity in MDA-MB-231 cells after LSD1 knockdown. Statistical analysis is performed using GraphPad Prism version 7.0. Means±s.d. is calculated by at least three independent experiments. * P
    Pcdna3 1 Slug Flag, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    CUL4B interacted with DDB1, RXB1 and DCAF11 to form a complex in vivo and in vitro . ( A ) Flag-tagged CUL4B interacted with RBX1 and DDB1 in vivo . The pCDNA3-CUL4B-Flag or pCDNA3-Flag empty vector was transformed into hFOB1.19, U2OS or Saos-2 cells. After 48 h incubation, Flag-tagged proteins were immunoprecipitated with anti-Flag agarose. The pull-down products were analysed by immunoblotting with anti-Flag, anti-RBX1, or anti-DDB1 antibodies. ( B ) Interactions between CUL4B/DDB1 and CUL4B/RBX1 in yeast. The pGBKT7-CUL4B plasmid was co-transformed with pGADT7-DDB1 or pGADT7-RBX1 into the yeast strain AH109. Growth of the transformed yeast was assayed on media lacking Trp and Leu (top panel) or lacking Trp, Leu, and His (bottom panel). Columns in each panel represent serial 10-fold dilutions. ( C ) Flag-tagged CUL4B associated with DCAF11 in vivo . The pCDNA3-CUL4B-Flag plasmid was transformed into hFOB1.19 (1), U2OS (2) or Saos-2 (3) cells. After 48 h incubation, Flag-tagged proteins were immunoprecipitated with either protein A agarose or anti-Flag-agarose. The cell lysate and pull-down products were analysed by immunoblotting with anti-CUL4B, anti-DCAF1, anti-DCAF4, anti-DCAF8, anti-DCAF11 or anti-DCAF15 antibodies. ( D ) DCAF11 was overexpressed in human osteosarcoma cell lines. Cell lysates were applied to WB analyses with specific antibodies against CUL4B, DDB1, RBX1, DCAF11, DCAF4, or β-Actin.

    Journal: Scientific Reports

    Article Title: CRL4BDCAF11 E3 ligase targets p21 for degradation to control cell cycle progression in human osteosarcoma cells

    doi: 10.1038/s41598-017-01344-9

    Figure Lengend Snippet: CUL4B interacted with DDB1, RXB1 and DCAF11 to form a complex in vivo and in vitro . ( A ) Flag-tagged CUL4B interacted with RBX1 and DDB1 in vivo . The pCDNA3-CUL4B-Flag or pCDNA3-Flag empty vector was transformed into hFOB1.19, U2OS or Saos-2 cells. After 48 h incubation, Flag-tagged proteins were immunoprecipitated with anti-Flag agarose. The pull-down products were analysed by immunoblotting with anti-Flag, anti-RBX1, or anti-DDB1 antibodies. ( B ) Interactions between CUL4B/DDB1 and CUL4B/RBX1 in yeast. The pGBKT7-CUL4B plasmid was co-transformed with pGADT7-DDB1 or pGADT7-RBX1 into the yeast strain AH109. Growth of the transformed yeast was assayed on media lacking Trp and Leu (top panel) or lacking Trp, Leu, and His (bottom panel). Columns in each panel represent serial 10-fold dilutions. ( C ) Flag-tagged CUL4B associated with DCAF11 in vivo . The pCDNA3-CUL4B-Flag plasmid was transformed into hFOB1.19 (1), U2OS (2) or Saos-2 (3) cells. After 48 h incubation, Flag-tagged proteins were immunoprecipitated with either protein A agarose or anti-Flag-agarose. The cell lysate and pull-down products were analysed by immunoblotting with anti-CUL4B, anti-DCAF1, anti-DCAF4, anti-DCAF8, anti-DCAF11 or anti-DCAF15 antibodies. ( D ) DCAF11 was overexpressed in human osteosarcoma cell lines. Cell lysates were applied to WB analyses with specific antibodies against CUL4B, DDB1, RBX1, DCAF11, DCAF4, or β-Actin.

    Article Snippet: Immunoprecipitation and mass spectrometry analysis Cells transfected with pCDNA3-Flag-HA-DCAF11 or pCDNA3-Flag-HA were lysed with Pierce IP lysis buffer (Thermo Fisher Scientific, USA) according to the manufacturer’s instructions and supplemented with 1x protease inhibitors (Roche, USA).

    Techniques: In Vivo, In Vitro, Plasmid Preparation, Transformation Assay, Incubation, Immunoprecipitation, Western Blot

    p21 is a target of CRL4B DCAF11 E3 ligase in human osteosarcoma cells. ( A ) Flag-tagged p21 associated with DCAF11 in vivo . The pCDNA3-p21-Flag plasmid was transformed into hFOB1.19 (1), U2OS (2) or Saos-2 (3) cells. After 48 h incubation, Flag-tagged proteins were immunoprecipitated with either protein A agarose or anti-Flag-agarose. The cell lysate and pull-down products were analysed by immunoblotting with anti-p21, anti-DCAF1, anti-DCAF4, anti-DCAF11 or β-Actin antibodies. ( B ) p21 interacted with DCAF11 in yeast. The pGBKT7-p21 plasmid was co-transformed with pGADT7-DCAF11 or pGADT7-DCAF4 into the yeast strain AH109. Growth of the transformed yeast was assayed on media lacking Trp and Leu (top panel) or lacking Trp, Leu, and His (bottom panel). Columns in each panel represent serial 10-fold dilutions. ( C ) p21 was significantly down-regulated in human osteosarcoma cell lines. Cell lysates were applied to WB analyses with specific antibodies against p21, CUL4B, p27, or β-Actin. ( D ) p21 was ubiquitinated by CRL4B DCAF11 E3 ligase in vitro . The purified His-p21 protein was incubated with E1, with or without E2, and with or without CUL4B-Flag in reaction buffer, followed by immunoblotting for p21.

    Journal: Scientific Reports

    Article Title: CRL4BDCAF11 E3 ligase targets p21 for degradation to control cell cycle progression in human osteosarcoma cells

    doi: 10.1038/s41598-017-01344-9

    Figure Lengend Snippet: p21 is a target of CRL4B DCAF11 E3 ligase in human osteosarcoma cells. ( A ) Flag-tagged p21 associated with DCAF11 in vivo . The pCDNA3-p21-Flag plasmid was transformed into hFOB1.19 (1), U2OS (2) or Saos-2 (3) cells. After 48 h incubation, Flag-tagged proteins were immunoprecipitated with either protein A agarose or anti-Flag-agarose. The cell lysate and pull-down products were analysed by immunoblotting with anti-p21, anti-DCAF1, anti-DCAF4, anti-DCAF11 or β-Actin antibodies. ( B ) p21 interacted with DCAF11 in yeast. The pGBKT7-p21 plasmid was co-transformed with pGADT7-DCAF11 or pGADT7-DCAF4 into the yeast strain AH109. Growth of the transformed yeast was assayed on media lacking Trp and Leu (top panel) or lacking Trp, Leu, and His (bottom panel). Columns in each panel represent serial 10-fold dilutions. ( C ) p21 was significantly down-regulated in human osteosarcoma cell lines. Cell lysates were applied to WB analyses with specific antibodies against p21, CUL4B, p27, or β-Actin. ( D ) p21 was ubiquitinated by CRL4B DCAF11 E3 ligase in vitro . The purified His-p21 protein was incubated with E1, with or without E2, and with or without CUL4B-Flag in reaction buffer, followed by immunoblotting for p21.

    Article Snippet: Immunoprecipitation and mass spectrometry analysis Cells transfected with pCDNA3-Flag-HA-DCAF11 or pCDNA3-Flag-HA were lysed with Pierce IP lysis buffer (Thermo Fisher Scientific, USA) according to the manufacturer’s instructions and supplemented with 1x protease inhibitors (Roche, USA).

    Techniques: In Vivo, Plasmid Preparation, Transformation Assay, Incubation, Immunoprecipitation, Western Blot, In Vitro, Purification

    The USP25 dimer stabilizes endogenous tankyrase in HEK293T cells. a Flag-USP25FL, Flag-USP25FL ΔIL, Flag-USP25FL C178A, Flag-USP25FL ΔIL C178A, Flag-USP25NCD, and Flag-USP25NCD ΔIL were transfected in HEK293T cells and the levels of endogenous tankyrases1/2 were analyzed by western blot (WB). GFP was transfected as a control. Plot of the quantification of tankyrase1/2 levels, corrected with tubulin and relative to GFP. Data values are mean ± s.d. and n ≥ 3 technical replicates. Significance was measured by a two-tailed unpaired t test for all lanes relative to GFP and between USP25FL and USP25FL ΔIL. * P

    Journal: Nature Communications

    Article Title: A quaternary tetramer assembly inhibits the deubiquitinating activity of USP25

    doi: 10.1038/s41467-018-07510-5

    Figure Lengend Snippet: The USP25 dimer stabilizes endogenous tankyrase in HEK293T cells. a Flag-USP25FL, Flag-USP25FL ΔIL, Flag-USP25FL C178A, Flag-USP25FL ΔIL C178A, Flag-USP25NCD, and Flag-USP25NCD ΔIL were transfected in HEK293T cells and the levels of endogenous tankyrases1/2 were analyzed by western blot (WB). GFP was transfected as a control. Plot of the quantification of tankyrase1/2 levels, corrected with tubulin and relative to GFP. Data values are mean ± s.d. and n ≥ 3 technical replicates. Significance was measured by a two-tailed unpaired t test for all lanes relative to GFP and between USP25FL and USP25FL ΔIL. * P

    Article Snippet: Cell culture vector constructs and transfection pcDNA3.1-Flag-USP25FL, pcDNA3.1-Flag-USP25FL ΔIL, pcDNA3.1-Flag-USP25FL C178A, pcDNA3.1-Flag-USP25FL ΔIL C178A, pcDNA3.1-Flag-USP25NCD, pcDNA3.1-Flag-USP25NCD ΔIL, and pcDNA3.1-Flag-USP25FL point mutants P521S, F522G, E373A, C651F, and L271W vector constructs were generated by PCR using previous pET28-Smt3 USP25 constructs as DNA templates, the specific primers fused with a Flag tag at the N Termini and Phusion DNA polymerase (Thermo) following the manufacturer’s instructions.

    Techniques: Transfection, Western Blot, Two Tailed Test

    Effect of mutations in the N-terminal region of the SBD on binding with PHD3. HEK293T cells were transfected with the expression plasmids for the proteins indicated at the top of each panel. The cells were lysed and the cell lysates were subjected to FLAG-immunoprecipiation (IP). Immunoprecipitates and lysates were then analyzed by western blotting using the indicated antibodies. ( a ) Both [S1-(P21H/A26P/Q62A)] NT [S2] CT and [S1-6Mut] NT [S2] CT chimeras only partially regained binding to PHD3, compared to binding of wild type Siah2 SBD to PHD3. ( b ) Only the chimera with the P21H/A26P mutations regained partial binding with PHD3. In contrast, mutation of Q62A did not increase PHD3 binding. FLAG-SBD Siah in the IP was masked by the IgG light chain.

    Journal: PLoS ONE

    Article Title: Investigating the Molecular Basis of Siah1 and Siah2 E3 Ubiquitin Ligase Substrate Specificity

    doi: 10.1371/journal.pone.0106547

    Figure Lengend Snippet: Effect of mutations in the N-terminal region of the SBD on binding with PHD3. HEK293T cells were transfected with the expression plasmids for the proteins indicated at the top of each panel. The cells were lysed and the cell lysates were subjected to FLAG-immunoprecipiation (IP). Immunoprecipitates and lysates were then analyzed by western blotting using the indicated antibodies. ( a ) Both [S1-(P21H/A26P/Q62A)] NT [S2] CT and [S1-6Mut] NT [S2] CT chimeras only partially regained binding to PHD3, compared to binding of wild type Siah2 SBD to PHD3. ( b ) Only the chimera with the P21H/A26P mutations regained partial binding with PHD3. In contrast, mutation of Q62A did not increase PHD3 binding. FLAG-SBD Siah in the IP was masked by the IgG light chain.

    Article Snippet: Plasmid constructs The pcDNA3.1 FLAG-SBD of human Siah2 (residues 130–392) and full-length (1–394) were constructed by PCR amplification of Siah2 cDNA fragments separately from the pCMV-SPORT6 plasmid (Thermo Scientific OpenBiosystems) with a Hind III-containing forward primer and an XbaI containing reverse primer.

    Techniques: Binding Assay, Transfection, Expressing, Western Blot, Mutagenesis

    Interaction of wild type (WT) and chimeric Siah proteins with PHD3. ( a ) Diagrammatic representation of the WT Siah1 and Siah2 SBD, which comprises of 1–193 residues, and the chimeric forms of Siah1 and Siah2 SBD, SBD[S1] NT [S2] CT and SBD[S2] NT [S1] CT . Corresponding original residue numbers are given in parentheses. ( b ) HEK293T cells were transfected in 60-mm cell culture plates for 2 days with the indicated expression plasmids. 48 hours after transfection, the cells were lysed and cell lysates were subjected to FLAG immunoprecipitation (IP). Immunopercipiates and the aliquotes of lysates were then immunoblotted using indicated antibodies. Both the chimeric forms lost binding to PHD3 as compared to wild type.

    Journal: PLoS ONE

    Article Title: Investigating the Molecular Basis of Siah1 and Siah2 E3 Ubiquitin Ligase Substrate Specificity

    doi: 10.1371/journal.pone.0106547

    Figure Lengend Snippet: Interaction of wild type (WT) and chimeric Siah proteins with PHD3. ( a ) Diagrammatic representation of the WT Siah1 and Siah2 SBD, which comprises of 1–193 residues, and the chimeric forms of Siah1 and Siah2 SBD, SBD[S1] NT [S2] CT and SBD[S2] NT [S1] CT . Corresponding original residue numbers are given in parentheses. ( b ) HEK293T cells were transfected in 60-mm cell culture plates for 2 days with the indicated expression plasmids. 48 hours after transfection, the cells were lysed and cell lysates were subjected to FLAG immunoprecipitation (IP). Immunopercipiates and the aliquotes of lysates were then immunoblotted using indicated antibodies. Both the chimeric forms lost binding to PHD3 as compared to wild type.

    Article Snippet: Plasmid constructs The pcDNA3.1 FLAG-SBD of human Siah2 (residues 130–392) and full-length (1–394) were constructed by PCR amplification of Siah2 cDNA fragments separately from the pCMV-SPORT6 plasmid (Thermo Scientific OpenBiosystems) with a Hind III-containing forward primer and an XbaI containing reverse primer.

    Techniques: Transfection, Cell Culture, Expressing, Immunoprecipitation, Binding Assay

    Effect of mutations in the C-terminal region of the SBD on binding with PHD3. HEK293T cells were transfected with the expression plasmids for the indicated proteins. The cells were lysed followed by FLAG immunoprecipitation (IP) of cell lysates. Immunoprecipitates and lysates were then analyzed by western blotting using the indicated antibodies. The [S2] NT [S1-Q121L] CT chimera regained binding equivalent to Siah2 SBD wild type. In contrast, [S2] NT [S1-T160A] CT showed only a small increase in PHD3 binding. FLAG-SBD Siah in the IP was masked by the IgG light chain.

    Journal: PLoS ONE

    Article Title: Investigating the Molecular Basis of Siah1 and Siah2 E3 Ubiquitin Ligase Substrate Specificity

    doi: 10.1371/journal.pone.0106547

    Figure Lengend Snippet: Effect of mutations in the C-terminal region of the SBD on binding with PHD3. HEK293T cells were transfected with the expression plasmids for the indicated proteins. The cells were lysed followed by FLAG immunoprecipitation (IP) of cell lysates. Immunoprecipitates and lysates were then analyzed by western blotting using the indicated antibodies. The [S2] NT [S1-Q121L] CT chimera regained binding equivalent to Siah2 SBD wild type. In contrast, [S2] NT [S1-T160A] CT showed only a small increase in PHD3 binding. FLAG-SBD Siah in the IP was masked by the IgG light chain.

    Article Snippet: Plasmid constructs The pcDNA3.1 FLAG-SBD of human Siah2 (residues 130–392) and full-length (1–394) were constructed by PCR amplification of Siah2 cDNA fragments separately from the pCMV-SPORT6 plasmid (Thermo Scientific OpenBiosystems) with a Hind III-containing forward primer and an XbaI containing reverse primer.

    Techniques: Binding Assay, Transfection, Expressing, Immunoprecipitation, Western Blot

    Siah1 exhibits weak binding compared to Siah2 with PHD3. HEK293T cells were transfected in 60-mm cell culture plates for 2 days with expression plasmids for the proteins indicated at the top of each panel. ( a ) Cell lysates were subjected to HA-IP and aliquots of the cell lysates and immunoprecipitates were analyzed by western blotting with the anti-FLAG antibody. Both the Full length and Siah1 SBD did not show binding to PHD3 ( b ) The lysates were subjected to reciprocal FLAG-IP. Immunoprecipitates and aliquots of the cell lysates were analyzed by Western blotting with anti-HA and anti-FLAG antibodies. In the IP, FLAG-SBD overlaps with the IgG light chain. Compared to Siah2 SBD, only weak binding of Siah1 SBD to PHD3 was observed.

    Journal: PLoS ONE

    Article Title: Investigating the Molecular Basis of Siah1 and Siah2 E3 Ubiquitin Ligase Substrate Specificity

    doi: 10.1371/journal.pone.0106547

    Figure Lengend Snippet: Siah1 exhibits weak binding compared to Siah2 with PHD3. HEK293T cells were transfected in 60-mm cell culture plates for 2 days with expression plasmids for the proteins indicated at the top of each panel. ( a ) Cell lysates were subjected to HA-IP and aliquots of the cell lysates and immunoprecipitates were analyzed by western blotting with the anti-FLAG antibody. Both the Full length and Siah1 SBD did not show binding to PHD3 ( b ) The lysates were subjected to reciprocal FLAG-IP. Immunoprecipitates and aliquots of the cell lysates were analyzed by Western blotting with anti-HA and anti-FLAG antibodies. In the IP, FLAG-SBD overlaps with the IgG light chain. Compared to Siah2 SBD, only weak binding of Siah1 SBD to PHD3 was observed.

    Article Snippet: Plasmid constructs The pcDNA3.1 FLAG-SBD of human Siah2 (residues 130–392) and full-length (1–394) were constructed by PCR amplification of Siah2 cDNA fragments separately from the pCMV-SPORT6 plasmid (Thermo Scientific OpenBiosystems) with a Hind III-containing forward primer and an XbaI containing reverse primer.

    Techniques: Binding Assay, Transfection, Cell Culture, Expressing, Western Blot

    Effect of mutations in Siah1 and Siah2 SBD Chimeras on binding with PHD3. ( a ) Pairwise sequence alignment of Siah1 and Siah2 SBD was performed by EMBOSS Needle tool. The 26 amino acids that are unique in Siah1 and Siah2 SBD are highlighted in grey. Dissimilar amino acids are highlighted by ‘*’. Similar amino acids are highlighted by ‘:’ and identical amino acids are highlighted by ‘|’ (top panel). The 10 dissimilar amino acids between Siah1 and Siah2 SBD are shown in diagrammatic representation of the chimeric forms, SBD[S1] NT [S2] CT and SBD[S1] NT [S2] CT (bottom panel). The original residue numbers are labeled in the respective colors ( b ) HEK293T cells were transfected with the indicated expression plasmids, followed by FLAG immunoprecipitation (IP) of cell lysates. Immunoprecipitates and lysates were then analyzed by western blotting using the indicated antibodies. The N-terminal mutant chimera, [S1-(E17S/P57S/F98H)] NT [S2] CT did not regain binding to PHD3 and the C-terminal mutant chimera, [S2] NT [S1-(Q121L/T160A] CT regained complete binding to PHD3 equivalent to WT Siah2 SBD.

    Journal: PLoS ONE

    Article Title: Investigating the Molecular Basis of Siah1 and Siah2 E3 Ubiquitin Ligase Substrate Specificity

    doi: 10.1371/journal.pone.0106547

    Figure Lengend Snippet: Effect of mutations in Siah1 and Siah2 SBD Chimeras on binding with PHD3. ( a ) Pairwise sequence alignment of Siah1 and Siah2 SBD was performed by EMBOSS Needle tool. The 26 amino acids that are unique in Siah1 and Siah2 SBD are highlighted in grey. Dissimilar amino acids are highlighted by ‘*’. Similar amino acids are highlighted by ‘:’ and identical amino acids are highlighted by ‘|’ (top panel). The 10 dissimilar amino acids between Siah1 and Siah2 SBD are shown in diagrammatic representation of the chimeric forms, SBD[S1] NT [S2] CT and SBD[S1] NT [S2] CT (bottom panel). The original residue numbers are labeled in the respective colors ( b ) HEK293T cells were transfected with the indicated expression plasmids, followed by FLAG immunoprecipitation (IP) of cell lysates. Immunoprecipitates and lysates were then analyzed by western blotting using the indicated antibodies. The N-terminal mutant chimera, [S1-(E17S/P57S/F98H)] NT [S2] CT did not regain binding to PHD3 and the C-terminal mutant chimera, [S2] NT [S1-(Q121L/T160A] CT regained complete binding to PHD3 equivalent to WT Siah2 SBD.

    Article Snippet: Plasmid constructs The pcDNA3.1 FLAG-SBD of human Siah2 (residues 130–392) and full-length (1–394) were constructed by PCR amplification of Siah2 cDNA fragments separately from the pCMV-SPORT6 plasmid (Thermo Scientific OpenBiosystems) with a Hind III-containing forward primer and an XbaI containing reverse primer.

    Techniques: Binding Assay, Sequencing, Labeling, Transfection, Expressing, Immunoprecipitation, Western Blot, Mutagenesis

    Effect of additional mutations in the N-terminal region of the SBD on binding with PHD3. ( a ) The 10 similar amino acids in the N terminal region (1–00) of Siah1 and Siah2 SBD are highlighted in grey. Mutated residues among the similar amino acids are highlighted within the box. ( b ) HEK293T cells were transfected with the expression plasmids for the indicated proteins. The cells were lysed and the cell lysates were subjected to FLAG-immunoprecipiation (IP). Immunoprecipitates and lysates were then analyzed by western blotting using the indicated antibodies. The [S1-8Mut] NT [S2] CT mutant increased binding compared to [S1-6Mut] NT [S2] CT . ( c ) The amount of PHD3 that coimmunoprecipitated with chimeric and mutated Siah1 and Siah2 SBD was quantified using Gel-pro analyzer software. The binding of the chimeric and mutated SBD to PHD3 was expressed as percentage of the binding of WT Siah2 SBD to PHD3. The data are represented as mean±S.E.M from three independent experiments. Differences in measured variables were assessed with Student's t test. * denotes p

    Journal: PLoS ONE

    Article Title: Investigating the Molecular Basis of Siah1 and Siah2 E3 Ubiquitin Ligase Substrate Specificity

    doi: 10.1371/journal.pone.0106547

    Figure Lengend Snippet: Effect of additional mutations in the N-terminal region of the SBD on binding with PHD3. ( a ) The 10 similar amino acids in the N terminal region (1–00) of Siah1 and Siah2 SBD are highlighted in grey. Mutated residues among the similar amino acids are highlighted within the box. ( b ) HEK293T cells were transfected with the expression plasmids for the indicated proteins. The cells were lysed and the cell lysates were subjected to FLAG-immunoprecipiation (IP). Immunoprecipitates and lysates were then analyzed by western blotting using the indicated antibodies. The [S1-8Mut] NT [S2] CT mutant increased binding compared to [S1-6Mut] NT [S2] CT . ( c ) The amount of PHD3 that coimmunoprecipitated with chimeric and mutated Siah1 and Siah2 SBD was quantified using Gel-pro analyzer software. The binding of the chimeric and mutated SBD to PHD3 was expressed as percentage of the binding of WT Siah2 SBD to PHD3. The data are represented as mean±S.E.M from three independent experiments. Differences in measured variables were assessed with Student's t test. * denotes p

    Article Snippet: Plasmid constructs The pcDNA3.1 FLAG-SBD of human Siah2 (residues 130–392) and full-length (1–394) were constructed by PCR amplification of Siah2 cDNA fragments separately from the pCMV-SPORT6 plasmid (Thermo Scientific OpenBiosystems) with a Hind III-containing forward primer and an XbaI containing reverse primer.

    Techniques: Binding Assay, Transfection, Expressing, Western Blot, Mutagenesis, Software

    Interaction of Siah2 with PHD3. ( a ) HEK293 cells were transfected in 60-mm cell culture plates for 2 days with the indicated expression plasmids. The cells were lysed, and the lysates were subjected to FLAG immunoprecipitation (IP), as described under “ Materials and Methods ”. Aliquots of the cell lysates and immunoprecipitates were analyzed by western blotting with the anti-HA antibody. Both full length Siah2 and Siah2 SBD bind to PHD3 to the same extent. In the IP, the presence of the faint band in the empty vector lane is due to non-specific binding of PHD3. The same membrane was reblotted with FLAG antibody to detect FLAG tagged Siah2 proteins. ( b ) GST-Siah2 SBD pulldown of HA-PHD3. Cell lysate of HEK293 cells transfected with HA-PHD3 was incubated with GST-Siah2 SBD immobilized on GSH agarose beads and the reaction was performed as described under “ Material and Methods ”. The empty expression vector alone was expressed as a GST control for non-specific binding of HA PHD3. After the incubation, the lysate was removed, the GSH-agarose beads were washed, and bound HA-PHD3 was analyzed by Western blotting using anti HA antibody. The pull down assay confirmed the interaction of Siah2 SBD with PHD3.

    Journal: PLoS ONE

    Article Title: Investigating the Molecular Basis of Siah1 and Siah2 E3 Ubiquitin Ligase Substrate Specificity

    doi: 10.1371/journal.pone.0106547

    Figure Lengend Snippet: Interaction of Siah2 with PHD3. ( a ) HEK293 cells were transfected in 60-mm cell culture plates for 2 days with the indicated expression plasmids. The cells were lysed, and the lysates were subjected to FLAG immunoprecipitation (IP), as described under “ Materials and Methods ”. Aliquots of the cell lysates and immunoprecipitates were analyzed by western blotting with the anti-HA antibody. Both full length Siah2 and Siah2 SBD bind to PHD3 to the same extent. In the IP, the presence of the faint band in the empty vector lane is due to non-specific binding of PHD3. The same membrane was reblotted with FLAG antibody to detect FLAG tagged Siah2 proteins. ( b ) GST-Siah2 SBD pulldown of HA-PHD3. Cell lysate of HEK293 cells transfected with HA-PHD3 was incubated with GST-Siah2 SBD immobilized on GSH agarose beads and the reaction was performed as described under “ Material and Methods ”. The empty expression vector alone was expressed as a GST control for non-specific binding of HA PHD3. After the incubation, the lysate was removed, the GSH-agarose beads were washed, and bound HA-PHD3 was analyzed by Western blotting using anti HA antibody. The pull down assay confirmed the interaction of Siah2 SBD with PHD3.

    Article Snippet: Plasmid constructs The pcDNA3.1 FLAG-SBD of human Siah2 (residues 130–392) and full-length (1–394) were constructed by PCR amplification of Siah2 cDNA fragments separately from the pCMV-SPORT6 plasmid (Thermo Scientific OpenBiosystems) with a Hind III-containing forward primer and an XbaI containing reverse primer.

    Techniques: Transfection, Cell Culture, Expressing, Immunoprecipitation, Western Blot, Plasmid Preparation, Binding Assay, Incubation, Pull Down Assay

    LSD1 acts cooperatively with Slug to inactivate ESR1 promoters by demethylating H3K4me2. ( a , b ) Western blot ( a ) and RT–PCR ( b ) analyze effects of LSD1 and Slug on ERα expression via Slug overexpression and LSD1 knockdown in MCF-7 cells. ( c , d ) Western blot ( c ) and RT–PCR ( d ) analysis of effects of LSD1 and Slug on ERα expression via Slug and LSD1 knockdown in MDA-MB-231 cells. ( e ) MDA-MB-231 cells expressing pHAGE-CMV-Flag-HA-LSD1 and pcDNA3.1-Slug-Flag or pcDNA3.1-ΔN-Slug-Flag was analyzed by IP. Cell lysates were immunoprecipitated with anti-HA antibody. Immunocomplexes were then immunoblotted using antibodies against Flag and HA. ( f ) A dual-luciferase reporter assay is used to investigate ESR1 promoter 2 activity in MDA-MB-231 cells by overexpressing wild-type or mutant Slug. ( g ) ChIP-qPCR analysis of LSD1 recruitment on ESR1 promoter in MDA-MB-231shSlug cells and their control cells. ( h ) ChIP-qPCR analysis of H3K4me2 recruitment on ESR1 promoter in MDA-MB-231shLSD1 cells and their control cells. All the ChIP-qPCR results represent % of input chromatin. ( i ) A dual-luciferase reporter assay is used to investigate ESR1 promoter activity in MDA-MB-231 cells after LSD1 knockdown. Statistical analysis is performed using GraphPad Prism version 7.0. Means±s.d. is calculated by at least three independent experiments. * P

    Journal: Oncogenesis

    Article Title: The zinc-finger transcriptional factor Slug transcriptionally downregulates ERα by recruiting lysine-specific demethylase 1 in human breast cancer

    doi: 10.1038/oncsis.2017.38

    Figure Lengend Snippet: LSD1 acts cooperatively with Slug to inactivate ESR1 promoters by demethylating H3K4me2. ( a , b ) Western blot ( a ) and RT–PCR ( b ) analyze effects of LSD1 and Slug on ERα expression via Slug overexpression and LSD1 knockdown in MCF-7 cells. ( c , d ) Western blot ( c ) and RT–PCR ( d ) analysis of effects of LSD1 and Slug on ERα expression via Slug and LSD1 knockdown in MDA-MB-231 cells. ( e ) MDA-MB-231 cells expressing pHAGE-CMV-Flag-HA-LSD1 and pcDNA3.1-Slug-Flag or pcDNA3.1-ΔN-Slug-Flag was analyzed by IP. Cell lysates were immunoprecipitated with anti-HA antibody. Immunocomplexes were then immunoblotted using antibodies against Flag and HA. ( f ) A dual-luciferase reporter assay is used to investigate ESR1 promoter 2 activity in MDA-MB-231 cells by overexpressing wild-type or mutant Slug. ( g ) ChIP-qPCR analysis of LSD1 recruitment on ESR1 promoter in MDA-MB-231shSlug cells and their control cells. ( h ) ChIP-qPCR analysis of H3K4me2 recruitment on ESR1 promoter in MDA-MB-231shLSD1 cells and their control cells. All the ChIP-qPCR results represent % of input chromatin. ( i ) A dual-luciferase reporter assay is used to investigate ESR1 promoter activity in MDA-MB-231 cells after LSD1 knockdown. Statistical analysis is performed using GraphPad Prism version 7.0. Means±s.d. is calculated by at least three independent experiments. * P

    Article Snippet: We transfected 4 μg (scaling up or down transfections based on plating medium volume) pcDNA3.1-Slug-Flag or pcDNA3.1 into MCF-7 or T47D cells growing in 60 mm tissue culture plates using Lipofectamine 2000 for 48 h. Stably transfected Slug MCF-7 cells or T47D were selected in DMEM with 10% FBS plus 1 mg/ml G418 (Thermo Fisher Scientific, Waltham, MA, USA).

    Techniques: Western Blot, Reverse Transcription Polymerase Chain Reaction, Expressing, Over Expression, Multiple Displacement Amplification, Immunoprecipitation, Luciferase, Reporter Assay, Activity Assay, Mutagenesis, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    Negative relationship between Slug and ERα in human breast cancer cells. ( a , b ) Analysis of Slug and ERα expression levels in MCF-7, T47D, SKBR3, MDA-MB-231 and BT549 by western blot and RT–PCR. ( c , d ) Western blot ( c ) and RT–PCR ( d ) analysis of protein and mRNA levels as indicated in MCF-7 cells transfected with increasing concentrations (2, 4 and 8 μg, respectively) of Slug. ( e , f ) Western blot ( e ) and RT–PCR ( f ) analysis of protein and mRNA levels in MDA-MB-231 cells transfected with two different interference sequences of Slug (40 n m , separately). ( g ) Western blot analysis of Slug knockdown level in stable cell lines of MDA-MB231shSlug. ( h ) Immunofluorescence micrographs of Slug (red) and ERα (green) expression in MDA-MB-231shNC cells (upper) and MDA-MB-231shSlug (lower). DAPI is for nuclei (blue). Magnification, × 400. Protein and mRNA expression was normalized to β-actin. In figure 2d, NC means transfecting with control pcDNA3.1. In figure 2e and f, siNC is scrambled control siRNA used for transient transfection. ShNC without shRNA is used as a control by infecting pLKO.1 lentivirus in MDA-MB-231 cell line. Statistical analysis is performed using GraphPad Prism version 7.0. Means±s.d. is calculated by at least three independent experiments. * P

    Journal: Oncogenesis

    Article Title: The zinc-finger transcriptional factor Slug transcriptionally downregulates ERα by recruiting lysine-specific demethylase 1 in human breast cancer

    doi: 10.1038/oncsis.2017.38

    Figure Lengend Snippet: Negative relationship between Slug and ERα in human breast cancer cells. ( a , b ) Analysis of Slug and ERα expression levels in MCF-7, T47D, SKBR3, MDA-MB-231 and BT549 by western blot and RT–PCR. ( c , d ) Western blot ( c ) and RT–PCR ( d ) analysis of protein and mRNA levels as indicated in MCF-7 cells transfected with increasing concentrations (2, 4 and 8 μg, respectively) of Slug. ( e , f ) Western blot ( e ) and RT–PCR ( f ) analysis of protein and mRNA levels in MDA-MB-231 cells transfected with two different interference sequences of Slug (40 n m , separately). ( g ) Western blot analysis of Slug knockdown level in stable cell lines of MDA-MB231shSlug. ( h ) Immunofluorescence micrographs of Slug (red) and ERα (green) expression in MDA-MB-231shNC cells (upper) and MDA-MB-231shSlug (lower). DAPI is for nuclei (blue). Magnification, × 400. Protein and mRNA expression was normalized to β-actin. In figure 2d, NC means transfecting with control pcDNA3.1. In figure 2e and f, siNC is scrambled control siRNA used for transient transfection. ShNC without shRNA is used as a control by infecting pLKO.1 lentivirus in MDA-MB-231 cell line. Statistical analysis is performed using GraphPad Prism version 7.0. Means±s.d. is calculated by at least three independent experiments. * P

    Article Snippet: We transfected 4 μg (scaling up or down transfections based on plating medium volume) pcDNA3.1-Slug-Flag or pcDNA3.1 into MCF-7 or T47D cells growing in 60 mm tissue culture plates using Lipofectamine 2000 for 48 h. Stably transfected Slug MCF-7 cells or T47D were selected in DMEM with 10% FBS plus 1 mg/ml G418 (Thermo Fisher Scientific, Waltham, MA, USA).

    Techniques: Expressing, Multiple Displacement Amplification, Western Blot, Reverse Transcription Polymerase Chain Reaction, Transfection, Stable Transfection, Immunofluorescence, shRNA