pegfp-n1 Search Results


  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 98
    Addgene inc pegfp n1
    Identification and validation of PTENβ. ( a ) The potential non-AUG initiation codons in favourable Kozak contexts in the 5′ UTR region of Homo sapiens PTEN mRNA are highlighted in red. The initiation codons of PTENα and PTEN are separately highlighted in yellow or green. ( b ) A <t>pEGFP-N1</t> plasmid containing PTENα with a C-terminal GFP tag was used for detection of PTENα, PTEN and unknown PTEN isoforms (upper panel); the indicated plasmids were introduced in HEK 293T cells followed by western blotting analysis using GFP antibody. β-tubulin was used as a control (lower panel). ( c ) pEGFP-N1 plasmids containing PTEN or PTENα with a C-terminal GFP tag, in which one of the two potential initiation codons (AUU 594 or UUG 621 ) was mutated to CUC combined with mutation of CUG 513 . ( d ) Mutation of AUU 594 but not UUG 621 eliminates PTENβ expression. Plasmids as indicated in c were introduced into HEK 293T cells separately, followed by immunoblotting with GFP antibody. ( e ) The Kozak context of AUU 594 and disruption of the Kozak context by site directed mutagenesis. ( f ) The Kozak context disruption abolishes PTENβ expression. HEK 293T cells were transfected with indicated constructs in e , followed by immunoblotting with GFP antibody. ( g ) A 12 bp AUU 594 downstream palindromic motif in the 5′ UTR of PTEN is evolutionarily conserved. Phylogenetic analysis of the 5′ UTR of PTEN mRNA in bonobo ( Pan paniscus ), killer whale ( Orcinus orca ) and mouse ( Mus musculus ). The alternative CUG 513 and AUU 594 codons are highlighted in blue boxes, and the 12 bp palindromic sequence is highlighted in a red box. ( h ) Disruption of the palindromic motif abolishes PTENβ expression. Upper panel, site directed mutagnesis was used to disrupt the AUU 594 downstream palindromic motif; lower panel, C-terminal GFP-tagged PTENβ expression plasmids with or without disruption of the palindromic motif were introduced into HEK 293T cells, followed by immunoblotting with GFP antibody.
    Pegfp N1, supplied by Addgene inc, used in various techniques. Bioz Stars score: 98/100, based on 204 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pegfp n1/product/Addgene inc
    Average 98 stars, based on 204 article reviews
    Price from $9.99 to $1999.99
    pegfp n1 - by Bioz Stars, 2020-04
    98/100 stars
      Buy from Supplier

    95
    Becton Dickinson pegfp n1
    (a) HLA-ABC and -DR surface expression in transiently transfected RPTECs. Cells were cotransfected with <t>pEGFP-N1</t> and either pCMV-agno or pCMV-ICP47 at a 1 : 10 ratio. pEGFP-N1 alone (mock, ▲) or together with pCMV-agno ( ♦ ) or with pCMV-ICP47 (□) as described above. MFI: mean fluorescence intensity. (b) HLA-ABC and -DR surface expression in transiently transfected UTA-6. UTA-6 cells were cotransfected with pEGFP-N1 and either pCMV-agno or pCMV-ICP47 at a 1 : 10 ratio. pEGFP-N1 alone (mock, ▲) or together with pCMV-agno ( ♦ ) or with pCMV-ICP47 (□) as described above. MFI: mean fluorescence intensity.
    Pegfp N1, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 95/100, based on 1010 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pegfp n1/product/Becton Dickinson
    Average 95 stars, based on 1010 article reviews
    Price from $9.99 to $1999.99
    pegfp n1 - by Bioz Stars, 2020-04
    95/100 stars
      Buy from Supplier

    99
    TaKaRa pegfp n1
    Localization of RyR in intracellular membranes of CHO cells. Confocal measurement of green fluorescence in CHO cells transfected with ( A ) <t>pEGFP-N1,</t> ( B ) pcDNA3 (GFP-RyR), and ( C ) pcDNA3 (GFP-RyR-C). Cells expressing GFP alone exhibit a diffuse pattern of fluorescence throughout the cell ( A ), whereas cells expressing the GFP-RyR and GFP-RyR-C fusion proteins show localized fluorescence signal near the perinuclear region of the cells ( B and C ). The panels from left to right show representative sections through the cells at 0.5-μm intervals. The cells have an average thickness of ∼10 μm.
    Pegfp N1, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 14900 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pegfp n1/product/TaKaRa
    Average 99 stars, based on 14900 article reviews
    Price from $9.99 to $1999.99
    pegfp n1 - by Bioz Stars, 2020-04
    99/100 stars
      Buy from Supplier

    94
    Thermo Fisher pegfp n1
    Efficient gene expression following electric pulse-mediated gene transfer. Plasmid DNA [65 μg of <t>pEGFP-N1</t> or 15 μg of Pgc-1 αL and 50 μg of plasmid DNA encoding an empty control vector (pCI-neo) or a mutant/deletion
    Pegfp N1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 1106 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pegfp n1/product/Thermo Fisher
    Average 94 stars, based on 1106 article reviews
    Price from $9.99 to $1999.99
    pegfp n1 - by Bioz Stars, 2020-04
    94/100 stars
      Buy from Supplier

    85
    SignaGen pegfp n1
    Efficient gene expression following electric pulse-mediated gene transfer. Plasmid DNA [65 μg of <t>pEGFP-N1</t> or 15 μg of Pgc-1 αL and 50 μg of plasmid DNA encoding an empty control vector (pCI-neo) or a mutant/deletion
    Pegfp N1, supplied by SignaGen, used in various techniques. Bioz Stars score: 85/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pegfp n1/product/SignaGen
    Average 85 stars, based on 8 article reviews
    Price from $9.99 to $1999.99
    pegfp n1 - by Bioz Stars, 2020-04
    85/100 stars
      Buy from Supplier

    87
    Elim Bio pegfp n1
    In-vitro analysis in IMR-90 cells. (a) MTT cell viability assay of the DNA/polymer complex with different polymers and at different DNA: polymer weight ratios in IMR-90. The amount of DNA was fixed at 1 μg). (b) Initial testing for PEV-L along with Lipofectamine 2000 (Lip) and PVBLG-8 (P0) with varying <t>pEGFP-N1</t> plasmid and polymer amount. Transfection efficiency was analysed 48 h post transfection with flow cytometry. (c) Fluorescent images of the transfection using Lip and PEV-H. Scale Bars = 0.25 mm.
    Pegfp N1, supplied by Elim Bio, used in various techniques. Bioz Stars score: 87/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pegfp n1/product/Elim Bio
    Average 87 stars, based on 7 article reviews
    Price from $9.99 to $1999.99
    pegfp n1 - by Bioz Stars, 2020-04
    87/100 stars
      Buy from Supplier

    93
    Promega pegfp n1
    In-vitro analysis in IMR-90 cells. (a) MTT cell viability assay of the DNA/polymer complex with different polymers and at different DNA: polymer weight ratios in IMR-90. The amount of DNA was fixed at 1 μg). (b) Initial testing for PEV-L along with Lipofectamine 2000 (Lip) and PVBLG-8 (P0) with varying <t>pEGFP-N1</t> plasmid and polymer amount. Transfection efficiency was analysed 48 h post transfection with flow cytometry. (c) Fluorescent images of the transfection using Lip and PEV-H. Scale Bars = 0.25 mm.
    Pegfp N1, supplied by Promega, used in various techniques. Bioz Stars score: 93/100, based on 140 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pegfp n1/product/Promega
    Average 93 stars, based on 140 article reviews
    Price from $9.99 to $1999.99
    pegfp n1 - by Bioz Stars, 2020-04
    93/100 stars
      Buy from Supplier

    92
    Stratagene pegfp n1
    Localization of VP4 expressed in COS-7 cells. COS-7 cells were transfected with <t>pEGFP-N1</t> (C) or pEGFP-N1-VP4 (A and B) and fixed at 48 h posttransfection, and the fluorescence was analyzed by confocal microscopy. Projection of all optical sections (A and C) and three optical sections located at the top, middle, and bottom of the transfected cell (B) are shown. Bar = 20 μm.
    Pegfp N1, supplied by Stratagene, used in various techniques. Bioz Stars score: 92/100, based on 120 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pegfp n1/product/Stratagene
    Average 92 stars, based on 120 article reviews
    Price from $9.99 to $1999.99
    pegfp n1 - by Bioz Stars, 2020-04
    92/100 stars
      Buy from Supplier

    93
    Millipore pegfp n1
    Localization of VP4 expressed in COS-7 cells. COS-7 cells were transfected with <t>pEGFP-N1</t> (C) or pEGFP-N1-VP4 (A and B) and fixed at 48 h posttransfection, and the fluorescence was analyzed by confocal microscopy. Projection of all optical sections (A and C) and three optical sections located at the top, middle, and bottom of the transfected cell (B) are shown. Bar = 20 μm.
    Pegfp N1, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 137 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pegfp n1/product/Millipore
    Average 93 stars, based on 137 article reviews
    Price from $9.99 to $1999.99
    pegfp n1 - by Bioz Stars, 2020-04
    93/100 stars
      Buy from Supplier

    86
    OriGene pegfp n1
    Specific targeting of scFv5T4. A) Histograms demonstrating surface binding of the indicated antibodies, either commercial mAb5T4 or generated scFv5T4 (empty curves), to 5T4 TAA on colorectal cancer cell line HT-29 measured by FACS. The shaded curves show the IgG2b isotype control (top panel) or streptavidin-FITC alone (bottom panel). B) Visualization of commercial mAb5T4, or scFv5T4, targeting of HEK293 cells transfected with empty vector (pCMV6-XL5) or pCMV6-XL5::5T4 by fluorescence microscopy. Representative images taken at 400× magnification. C) Visualization of HEK293 transfected with <t>pEGFP-N1</t> or pEGFP-N1::5T4 and incubated with scFv5T4::mRFP1. Same field of view photographs were taken under phase contrast, and green and red fluorescent filters at 100× magnification.
    Pegfp N1, supplied by OriGene, used in various techniques. Bioz Stars score: 86/100, based on 36 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pegfp n1/product/OriGene
    Average 86 stars, based on 36 article reviews
    Price from $9.99 to $1999.99
    pegfp n1 - by Bioz Stars, 2020-04
    86/100 stars
      Buy from Supplier

    94
    TaKaRa control pegfp n1
    Specific targeting of scFv5T4. A) Histograms demonstrating surface binding of the indicated antibodies, either commercial mAb5T4 or generated scFv5T4 (empty curves), to 5T4 TAA on colorectal cancer cell line HT-29 measured by FACS. The shaded curves show the IgG2b isotype control (top panel) or streptavidin-FITC alone (bottom panel). B) Visualization of commercial mAb5T4, or scFv5T4, targeting of HEK293 cells transfected with empty vector (pCMV6-XL5) or pCMV6-XL5::5T4 by fluorescence microscopy. Representative images taken at 400× magnification. C) Visualization of HEK293 transfected with <t>pEGFP-N1</t> or pEGFP-N1::5T4 and incubated with scFv5T4::mRFP1. Same field of view photographs were taken under phase contrast, and green and red fluorescent filters at 100× magnification.
    Control Pegfp N1, supplied by TaKaRa, used in various techniques. Bioz Stars score: 94/100, based on 24 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/control pegfp n1/product/TaKaRa
    Average 94 stars, based on 24 article reviews
    Price from $9.99 to $1999.99
    control pegfp n1 - by Bioz Stars, 2020-04
    94/100 stars
      Buy from Supplier

    94
    Addgene inc pegfp n1 tfeb
    ApoA-I Iowa fibrils were degraded via the autophagy-lysosomal pathway. ( a ) HEK293 cells were plated on poly-L-lysine-coated cover glasses and treated with apoA-I Iowa fibrils (1 μM) at 37 °C for 12 h. Cells were washed with fresh DMEM and then incubated in DMEM at 37 °C for 0, 6, 12, or 24 h. ApoA-I Iowa fibrils were visualized by using an anti-apoA-I antibody and an Alexa Fluor 568-conjugated secondary antibody. ( b ) HEK293 cells were treated with apoA-I Iowa fibrils (1 μM) at 37 °C for 12 h. Cells were washed with fresh DMEM and then incubated in DMEM for 6 h, after which whole cell lysates were prepared. The apoA-I contents of the whole cell lysates were analyzed by means of dot blotting. β-Actin was used as a loading control. The graph shows quantification of cellular apoA-I Iowa fibrils. Data are means ± SE of three independent experiments. ( c , d , e ) HEK293 cells were plated and treated with 1 μM apoA-I Iowa fibrils for 12 h. Cells were washed with fresh DMEM and cultured at 37 °C for an additional 6 or 12 h in fresh DMEM in the presence or absence of chloroquine ( c ; 50 μg/mL, 12 h), 3-MA ( d ; 0.75 μg/mL, 12 h), or rapamycin (e; 2.5 μM, 6 h), after which whole cell lysates were prepared. The apoA-I contents of the whole cell lysates were analyzed by using dot blotting. β-Actin was used as a loading control. ( f ) Effects of <t>TFEB</t> overexpression on cellular degradation of apoA-I Iowa fibrils. HEK293 cells were plated, transfected with <t>pEGFP-N1-TFEB</t> or an empty vector, and cultured at 37 °C for 48 h. Cells were treated with 1 μM apoA-I Iowa fibrils for 12 h, washed with fresh DMEM, and incubated for 12 h, after which whole cell lysates were prepared. The apoA-I contents in whole cell lysates were analyzed by dot blotting with an anti-apoA-I antibody. β-Actin was used as a loading control. The graphs show quantification of the degraded fibrils. Data are means ± SE of three independent experiments. * p = 0.0013 ( c ), 0.011 ( d ), 0.017 ( e ), and 0.032 ( f ) versus each drug or TFEB (−) cells.
    Pegfp N1 Tfeb, supplied by Addgene inc, used in various techniques. Bioz Stars score: 94/100, based on 112 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pegfp n1 tfeb/product/Addgene inc
    Average 94 stars, based on 112 article reviews
    Price from $9.99 to $1999.99
    pegfp n1 tfeb - by Bioz Stars, 2020-04
    94/100 stars
      Buy from Supplier

    93
    Addgene inc empty pegfp n1
    ApoA-I Iowa fibrils were degraded via the autophagy-lysosomal pathway. ( a ) HEK293 cells were plated on poly-L-lysine-coated cover glasses and treated with apoA-I Iowa fibrils (1 μM) at 37 °C for 12 h. Cells were washed with fresh DMEM and then incubated in DMEM at 37 °C for 0, 6, 12, or 24 h. ApoA-I Iowa fibrils were visualized by using an anti-apoA-I antibody and an Alexa Fluor 568-conjugated secondary antibody. ( b ) HEK293 cells were treated with apoA-I Iowa fibrils (1 μM) at 37 °C for 12 h. Cells were washed with fresh DMEM and then incubated in DMEM for 6 h, after which whole cell lysates were prepared. The apoA-I contents of the whole cell lysates were analyzed by means of dot blotting. β-Actin was used as a loading control. The graph shows quantification of cellular apoA-I Iowa fibrils. Data are means ± SE of three independent experiments. ( c , d , e ) HEK293 cells were plated and treated with 1 μM apoA-I Iowa fibrils for 12 h. Cells were washed with fresh DMEM and cultured at 37 °C for an additional 6 or 12 h in fresh DMEM in the presence or absence of chloroquine ( c ; 50 μg/mL, 12 h), 3-MA ( d ; 0.75 μg/mL, 12 h), or rapamycin (e; 2.5 μM, 6 h), after which whole cell lysates were prepared. The apoA-I contents of the whole cell lysates were analyzed by using dot blotting. β-Actin was used as a loading control. ( f ) Effects of <t>TFEB</t> overexpression on cellular degradation of apoA-I Iowa fibrils. HEK293 cells were plated, transfected with <t>pEGFP-N1-TFEB</t> or an empty vector, and cultured at 37 °C for 48 h. Cells were treated with 1 μM apoA-I Iowa fibrils for 12 h, washed with fresh DMEM, and incubated for 12 h, after which whole cell lysates were prepared. The apoA-I contents in whole cell lysates were analyzed by dot blotting with an anti-apoA-I antibody. β-Actin was used as a loading control. The graphs show quantification of the degraded fibrils. Data are means ± SE of three independent experiments. * p = 0.0013 ( c ), 0.011 ( d ), 0.017 ( e ), and 0.032 ( f ) versus each drug or TFEB (−) cells.
    Empty Pegfp N1, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/empty pegfp n1/product/Addgene inc
    Average 93 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    empty pegfp n1 - by Bioz Stars, 2020-04
    93/100 stars
      Buy from Supplier

    91
    Addgene inc pegfp n1 hdac6
    ApoA-I Iowa fibrils were degraded via the autophagy-lysosomal pathway. ( a ) HEK293 cells were plated on poly-L-lysine-coated cover glasses and treated with apoA-I Iowa fibrils (1 μM) at 37 °C for 12 h. Cells were washed with fresh DMEM and then incubated in DMEM at 37 °C for 0, 6, 12, or 24 h. ApoA-I Iowa fibrils were visualized by using an anti-apoA-I antibody and an Alexa Fluor 568-conjugated secondary antibody. ( b ) HEK293 cells were treated with apoA-I Iowa fibrils (1 μM) at 37 °C for 12 h. Cells were washed with fresh DMEM and then incubated in DMEM for 6 h, after which whole cell lysates were prepared. The apoA-I contents of the whole cell lysates were analyzed by means of dot blotting. β-Actin was used as a loading control. The graph shows quantification of cellular apoA-I Iowa fibrils. Data are means ± SE of three independent experiments. ( c , d , e ) HEK293 cells were plated and treated with 1 μM apoA-I Iowa fibrils for 12 h. Cells were washed with fresh DMEM and cultured at 37 °C for an additional 6 or 12 h in fresh DMEM in the presence or absence of chloroquine ( c ; 50 μg/mL, 12 h), 3-MA ( d ; 0.75 μg/mL, 12 h), or rapamycin (e; 2.5 μM, 6 h), after which whole cell lysates were prepared. The apoA-I contents of the whole cell lysates were analyzed by using dot blotting. β-Actin was used as a loading control. ( f ) Effects of <t>TFEB</t> overexpression on cellular degradation of apoA-I Iowa fibrils. HEK293 cells were plated, transfected with <t>pEGFP-N1-TFEB</t> or an empty vector, and cultured at 37 °C for 48 h. Cells were treated with 1 μM apoA-I Iowa fibrils for 12 h, washed with fresh DMEM, and incubated for 12 h, after which whole cell lysates were prepared. The apoA-I contents in whole cell lysates were analyzed by dot blotting with an anti-apoA-I antibody. β-Actin was used as a loading control. The graphs show quantification of the degraded fibrils. Data are means ± SE of three independent experiments. * p = 0.0013 ( c ), 0.011 ( d ), 0.017 ( e ), and 0.032 ( f ) versus each drug or TFEB (−) cells.
    Pegfp N1 Hdac6, supplied by Addgene inc, used in various techniques. Bioz Stars score: 91/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pegfp n1 hdac6/product/Addgene inc
    Average 91 stars, based on 5 article reviews
    Price from $9.99 to $1999.99
    pegfp n1 hdac6 - by Bioz Stars, 2020-04
    91/100 stars
      Buy from Supplier

    93
    Addgene inc pegfp n1 flag
    ApoA-I Iowa fibrils were degraded via the autophagy-lysosomal pathway. ( a ) HEK293 cells were plated on poly-L-lysine-coated cover glasses and treated with apoA-I Iowa fibrils (1 μM) at 37 °C for 12 h. Cells were washed with fresh DMEM and then incubated in DMEM at 37 °C for 0, 6, 12, or 24 h. ApoA-I Iowa fibrils were visualized by using an anti-apoA-I antibody and an Alexa Fluor 568-conjugated secondary antibody. ( b ) HEK293 cells were treated with apoA-I Iowa fibrils (1 μM) at 37 °C for 12 h. Cells were washed with fresh DMEM and then incubated in DMEM for 6 h, after which whole cell lysates were prepared. The apoA-I contents of the whole cell lysates were analyzed by means of dot blotting. β-Actin was used as a loading control. The graph shows quantification of cellular apoA-I Iowa fibrils. Data are means ± SE of three independent experiments. ( c , d , e ) HEK293 cells were plated and treated with 1 μM apoA-I Iowa fibrils for 12 h. Cells were washed with fresh DMEM and cultured at 37 °C for an additional 6 or 12 h in fresh DMEM in the presence or absence of chloroquine ( c ; 50 μg/mL, 12 h), 3-MA ( d ; 0.75 μg/mL, 12 h), or rapamycin (e; 2.5 μM, 6 h), after which whole cell lysates were prepared. The apoA-I contents of the whole cell lysates were analyzed by using dot blotting. β-Actin was used as a loading control. ( f ) Effects of <t>TFEB</t> overexpression on cellular degradation of apoA-I Iowa fibrils. HEK293 cells were plated, transfected with <t>pEGFP-N1-TFEB</t> or an empty vector, and cultured at 37 °C for 48 h. Cells were treated with 1 μM apoA-I Iowa fibrils for 12 h, washed with fresh DMEM, and incubated for 12 h, after which whole cell lysates were prepared. The apoA-I contents in whole cell lysates were analyzed by dot blotting with an anti-apoA-I antibody. β-Actin was used as a loading control. The graphs show quantification of the degraded fibrils. Data are means ± SE of three independent experiments. * p = 0.0013 ( c ), 0.011 ( d ), 0.017 ( e ), and 0.032 ( f ) versus each drug or TFEB (−) cells.
    Pegfp N1 Flag, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pegfp n1 flag/product/Addgene inc
    Average 93 stars, based on 11 article reviews
    Price from $9.99 to $1999.99
    pegfp n1 flag - by Bioz Stars, 2020-04
    93/100 stars
      Buy from Supplier

    88
    Becton Dickinson plasmid pegfp n1
    Laforin is phosphorylated at residue Ser25. A) Cell extracts from HEK293 cells transfected with plasmids pCMVmyc-laforin (WT, S25D and T194I) were analyzed by 2D-electrophoresis and western blotting using anti-myc monoclonal antibodies. Cell extracts were also treated with λ-phosphatase as described in Experimental section. The calculated pI for each spot is indicated. Molecular size standards are indicated in the right. B) Cell extracts from HEK293 cells co-transfected with plasmids pCMVmyclaforin (WT, S25A and S25D) and <t>pEGFP-N1</t> (expressing GFP), were prepared for immunodetection as described in Experimental section. In this case, lysis buffer contained 0.5% TritonX100 instead of 0.5% nonidet P40. Extracts were centrifuged at 13,000 rpm for 10 min at 4°C to separate soluble from pellet fractions. These fractions were treated with SDS-PAGE sample buffer and analyzed by western blotting using anti-myc and anti-GFP antibodies. C) HEK293 cells co-transfected with plasmids pCMVmyc-laforin (WT, S25A and S25D) were treated or not with 10 μM MG132 (a proteasome inhibitor) for 8 hours. Then, crude extracts were obtained and analyzed by western blotting using anti-myc and anti-tubulin (loading control).
    Plasmid Pegfp N1, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 88/100, based on 107 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/plasmid pegfp n1/product/Becton Dickinson
    Average 88 stars, based on 107 article reviews
    Price from $9.99 to $1999.99
    plasmid pegfp n1 - by Bioz Stars, 2020-04
    88/100 stars
      Buy from Supplier

    85
    TaKaRa fluorescent protein pegfp n1
    Transfection efficiencies mediated by RTNs carrying <t>pEGFP</t> plasmid and assessed by flow cytometry. RTNs were formed using <t>pEGFP-N1</t> plasmid DNA. Aliquots of nanocomplexes nebulised through the NGI (as in Figure 2A and 3 ) were used to perform transfections. After 48 h incubation, cells were harvested and analysed by flow cytometry to determine the level of expression of enhanced green fluorescent protein (EGFP) in the samples collected from each NGI stage. A representative experiment carried out in normal human bronchial epithelial cell-line 16HBE14o- is shown (cumulative experiments are summarised in Table 2 ). The dot-plot profile of untransfected cells (ctrl) represents the population selected as alive whereas the 1st panel of histogram profile shows where the markers to discriminate the EGFP positive from negative population were set. On each panel the percentage of EGFP negative (left) and EGFP positive (right) cells is indicated. PN = represent cells transfected with pre-nebulisation RTN suspension; LO = cells treated with leftover nanocomplexes in the nebuliser chamber after nebulisation, Thr = throat, and S1–S8 cells transfected with samples from the different NGI stages, respectively.
    Fluorescent Protein Pegfp N1, supplied by TaKaRa, used in various techniques. Bioz Stars score: 85/100, based on 44 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/fluorescent protein pegfp n1/product/TaKaRa
    Average 85 stars, based on 44 article reviews
    Price from $9.99 to $1999.99
    fluorescent protein pegfp n1 - by Bioz Stars, 2020-04
    85/100 stars
      Buy from Supplier

    95
    Beyotime pegfp n1 vector
    Transfection efficiencies mediated by RTNs carrying <t>pEGFP</t> plasmid and assessed by flow cytometry. RTNs were formed using <t>pEGFP-N1</t> plasmid DNA. Aliquots of nanocomplexes nebulised through the NGI (as in Figure 2A and 3 ) were used to perform transfections. After 48 h incubation, cells were harvested and analysed by flow cytometry to determine the level of expression of enhanced green fluorescent protein (EGFP) in the samples collected from each NGI stage. A representative experiment carried out in normal human bronchial epithelial cell-line 16HBE14o- is shown (cumulative experiments are summarised in Table 2 ). The dot-plot profile of untransfected cells (ctrl) represents the population selected as alive whereas the 1st panel of histogram profile shows where the markers to discriminate the EGFP positive from negative population were set. On each panel the percentage of EGFP negative (left) and EGFP positive (right) cells is indicated. PN = represent cells transfected with pre-nebulisation RTN suspension; LO = cells treated with leftover nanocomplexes in the nebuliser chamber after nebulisation, Thr = throat, and S1–S8 cells transfected with samples from the different NGI stages, respectively.
    Pegfp N1 Vector, supplied by Beyotime, used in various techniques. Bioz Stars score: 95/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pegfp n1 vector/product/Beyotime
    Average 95 stars, based on 16 article reviews
    Price from $9.99 to $1999.99
    pegfp n1 vector - by Bioz Stars, 2020-04
    95/100 stars
      Buy from Supplier

    95
    Promega pegfp n1 vector
    HCCR-1 is localized on the mitochondria outer membrane . (A) COS-7 (top and second panels), MCF-7 (third and forth panels) and HEK293/HCCR-1-V5 (bottom panel) cells were cultured on the pre-coated coverslip for overnight. COS-7 and MCF-7 cells were transiently transfected with the <t>pEGFP-N1</t> vector (GFP) or the vector carrying the human HCCR-1 gene tagged with GFP at its COOH-terminus (HCCR-1/GFP). The cells were incubated with MitoTracker (Red), fixed with 4% paraformaldyhyde. HEK293/HCCR-1-V5 cells were stained with MitoTracker (Red) and fixed with 100% methanol for 1 min at -20°C. Mouse anti-V5 antibody (1:300 dilution) and goat anti-mouse IgG conjugated with HRP (5 μg/ml) were applied to the cells for 1 h at room temperature (HCCR-1-V5). The cells were mounted and examined using a confocal microscope. (B) HCCR-1 is expressed at the mitochondrial membrane in the HEK293 cell line. Subcellular fraction of HEK293/HCCR-1-V5 cell line. HEK293/HCCR-1-V5 cells were fractionated into the nuclei (lane 1), post-mitochondria (lane 2), and mitochondria (lane3) fractions by differential centrifugation. Isolated mitochondria were treated with 0.1 M Na 2 CO 3 and fractionated by centrifugation at 100,000 g into pellets (lane 4) and supernatant (lane 5). Proteins were analyzed by immunoblotting using antibodies to the V5 epitope (top panel), the mitochondrial membrane protein VDAC-1 (middle panel) and the mitochondrial matrix protein SOD-2 (bottom panel). (C) HCCR-1 topology on the mitochondrial outer membrane. Intact mitochondria were incubated for 30 min at room temperature without (lane 1) and with 50 μg/ml proteinase K (lane 2) or with 50 μg/ml proteinase K plus 1% Triton X-100 (lane 3). Each sample was precipitated with TCA and separated by SDS-PAGE and analyzed by immunoblotting with antibodies to V5 (top panel), to the outer membrane protein TOM40 (second panel) and to the inner membrane protein TIM23 (third panel), and to the matrix protein SOD-2 (bottom panel). (D) Schematic diagram shows the HCCR-1 topology at the outer mitochondrial membrane.
    Pegfp N1 Vector, supplied by Promega, used in various techniques. Bioz Stars score: 95/100, based on 85 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pegfp n1 vector/product/Promega
    Average 95 stars, based on 85 article reviews
    Price from $9.99 to $1999.99
    pegfp n1 vector - by Bioz Stars, 2020-04
    95/100 stars
      Buy from Supplier

    93
    Thermo Fisher pegfp n1 vector
    Western blot ( IB ) analysis of heterodimerization of α 2B -AR and M6A or Y12A/S13A mutants A, dimerization of α 2B -ARand M6A. HEK293T cells were transfected with HA-tagged α 2B -AR plus the <t>pEGFP-N1</t> vector (control), GFP-tagged α 2B -AR (WT) or GFP-tagged M6A. IP, immunoprecipitation. B, dimerization of α 2B -AR and Y12A/S13A. HEK293T cells were transfected with HA- α 2B -AR together with the pEGFP-N1 vector (control), GFP-tagged α 2B -AR (WT) or GFP-tagged Y12A/S13A. The cells were solubilized and immu-noprecipitated with anti-GFP antibodies. The immunoprecipitate was sepa-rated by SDS-PAGE, and immunoprecipitated receptors were revealed by Western blotting with anti-HAantibodies ( upper panels). The blots were then stripedand reprobed with anti-GFP antibodies ( lower panels). The data shown in A and B are representative of three independent experiments.
    Pegfp N1 Vector, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 390 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pegfp n1 vector/product/Thermo Fisher
    Average 93 stars, based on 390 article reviews
    Price from $9.99 to $1999.99
    pegfp n1 vector - by Bioz Stars, 2020-04
    93/100 stars
      Buy from Supplier

    92
    Addgene inc vector pegfp n1
    Western blot ( IB ) analysis of heterodimerization of α 2B -AR and M6A or Y12A/S13A mutants A, dimerization of α 2B -ARand M6A. HEK293T cells were transfected with HA-tagged α 2B -AR plus the <t>pEGFP-N1</t> vector (control), GFP-tagged α 2B -AR (WT) or GFP-tagged M6A. IP, immunoprecipitation. B, dimerization of α 2B -AR and Y12A/S13A. HEK293T cells were transfected with HA- α 2B -AR together with the pEGFP-N1 vector (control), GFP-tagged α 2B -AR (WT) or GFP-tagged Y12A/S13A. The cells were solubilized and immu-noprecipitated with anti-GFP antibodies. The immunoprecipitate was sepa-rated by SDS-PAGE, and immunoprecipitated receptors were revealed by Western blotting with anti-HAantibodies ( upper panels). The blots were then stripedand reprobed with anti-GFP antibodies ( lower panels). The data shown in A and B are representative of three independent experiments.
    Vector Pegfp N1, supplied by Addgene inc, used in various techniques. Bioz Stars score: 92/100, based on 27 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/vector pegfp n1/product/Addgene inc
    Average 92 stars, based on 27 article reviews
    Price from $9.99 to $1999.99
    vector pegfp n1 - by Bioz Stars, 2020-04
    92/100 stars
      Buy from Supplier

    92
    Beijing Solarbio Science reagents pegfp n1
    Western blot ( IB ) analysis of heterodimerization of α 2B -AR and M6A or Y12A/S13A mutants A, dimerization of α 2B -ARand M6A. HEK293T cells were transfected with HA-tagged α 2B -AR plus the <t>pEGFP-N1</t> vector (control), GFP-tagged α 2B -AR (WT) or GFP-tagged M6A. IP, immunoprecipitation. B, dimerization of α 2B -AR and Y12A/S13A. HEK293T cells were transfected with HA- α 2B -AR together with the pEGFP-N1 vector (control), GFP-tagged α 2B -AR (WT) or GFP-tagged Y12A/S13A. The cells were solubilized and immu-noprecipitated with anti-GFP antibodies. The immunoprecipitate was sepa-rated by SDS-PAGE, and immunoprecipitated receptors were revealed by Western blotting with anti-HAantibodies ( upper panels). The blots were then stripedand reprobed with anti-GFP antibodies ( lower panels). The data shown in A and B are representative of three independent experiments.
    Reagents Pegfp N1, supplied by Beijing Solarbio Science, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/reagents pegfp n1/product/Beijing Solarbio Science
    Average 92 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    reagents pegfp n1 - by Bioz Stars, 2020-04
    92/100 stars
      Buy from Supplier

    91
    Genechem pegfp n1 plasmid
    Western blot ( IB ) analysis of heterodimerization of α 2B -AR and M6A or Y12A/S13A mutants A, dimerization of α 2B -ARand M6A. HEK293T cells were transfected with HA-tagged α 2B -AR plus the <t>pEGFP-N1</t> vector (control), GFP-tagged α 2B -AR (WT) or GFP-tagged M6A. IP, immunoprecipitation. B, dimerization of α 2B -AR and Y12A/S13A. HEK293T cells were transfected with HA- α 2B -AR together with the pEGFP-N1 vector (control), GFP-tagged α 2B -AR (WT) or GFP-tagged Y12A/S13A. The cells were solubilized and immu-noprecipitated with anti-GFP antibodies. The immunoprecipitate was sepa-rated by SDS-PAGE, and immunoprecipitated receptors were revealed by Western blotting with anti-HAantibodies ( upper panels). The blots were then stripedand reprobed with anti-GFP antibodies ( lower panels). The data shown in A and B are representative of three independent experiments.
    Pegfp N1 Plasmid, supplied by Genechem, used in various techniques. Bioz Stars score: 91/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pegfp n1 plasmid/product/Genechem
    Average 91 stars, based on 12 article reviews
    Price from $9.99 to $1999.99
    pegfp n1 plasmid - by Bioz Stars, 2020-04
    91/100 stars
      Buy from Supplier

    90
    Addgene inc pegfp n1 mek1
    Western blot ( IB ) analysis of heterodimerization of α 2B -AR and M6A or Y12A/S13A mutants A, dimerization of α 2B -ARand M6A. HEK293T cells were transfected with HA-tagged α 2B -AR plus the <t>pEGFP-N1</t> vector (control), GFP-tagged α 2B -AR (WT) or GFP-tagged M6A. IP, immunoprecipitation. B, dimerization of α 2B -AR and Y12A/S13A. HEK293T cells were transfected with HA- α 2B -AR together with the pEGFP-N1 vector (control), GFP-tagged α 2B -AR (WT) or GFP-tagged Y12A/S13A. The cells were solubilized and immu-noprecipitated with anti-GFP antibodies. The immunoprecipitate was sepa-rated by SDS-PAGE, and immunoprecipitated receptors were revealed by Western blotting with anti-HAantibodies ( upper panels). The blots were then stripedand reprobed with anti-GFP antibodies ( lower panels). The data shown in A and B are representative of three independent experiments.
    Pegfp N1 Mek1, supplied by Addgene inc, used in various techniques. Bioz Stars score: 90/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pegfp n1 mek1/product/Addgene inc
    Average 90 stars, based on 8 article reviews
    Price from $9.99 to $1999.99
    pegfp n1 mek1 - by Bioz Stars, 2020-04
    90/100 stars
      Buy from Supplier

    85
    Addgene inc pegfp n1 q61l rac
    Western blot ( IB ) analysis of heterodimerization of α 2B -AR and M6A or Y12A/S13A mutants A, dimerization of α 2B -ARand M6A. HEK293T cells were transfected with HA-tagged α 2B -AR plus the <t>pEGFP-N1</t> vector (control), GFP-tagged α 2B -AR (WT) or GFP-tagged M6A. IP, immunoprecipitation. B, dimerization of α 2B -AR and Y12A/S13A. HEK293T cells were transfected with HA- α 2B -AR together with the pEGFP-N1 vector (control), GFP-tagged α 2B -AR (WT) or GFP-tagged Y12A/S13A. The cells were solubilized and immu-noprecipitated with anti-GFP antibodies. The immunoprecipitate was sepa-rated by SDS-PAGE, and immunoprecipitated receptors were revealed by Western blotting with anti-HAantibodies ( upper panels). The blots were then stripedand reprobed with anti-GFP antibodies ( lower panels). The data shown in A and B are representative of three independent experiments.
    Pegfp N1 Q61l Rac, supplied by Addgene inc, used in various techniques. Bioz Stars score: 85/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pegfp n1 q61l rac/product/Addgene inc
    Average 85 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    pegfp n1 q61l rac - by Bioz Stars, 2020-04
    85/100 stars
      Buy from Supplier

    84
    of South Carolina pegfp n1
    Western blot ( IB ) analysis of heterodimerization of α 2B -AR and M6A or Y12A/S13A mutants A, dimerization of α 2B -ARand M6A. HEK293T cells were transfected with HA-tagged α 2B -AR plus the <t>pEGFP-N1</t> vector (control), GFP-tagged α 2B -AR (WT) or GFP-tagged M6A. IP, immunoprecipitation. B, dimerization of α 2B -AR and Y12A/S13A. HEK293T cells were transfected with HA- α 2B -AR together with the pEGFP-N1 vector (control), GFP-tagged α 2B -AR (WT) or GFP-tagged Y12A/S13A. The cells were solubilized and immu-noprecipitated with anti-GFP antibodies. The immunoprecipitate was sepa-rated by SDS-PAGE, and immunoprecipitated receptors were revealed by Western blotting with anti-HAantibodies ( upper panels). The blots were then stripedand reprobed with anti-GFP antibodies ( lower panels). The data shown in A and B are representative of three independent experiments.
    Pegfp N1, supplied by of South Carolina, used in various techniques. Bioz Stars score: 84/100, based on 28 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pegfp n1/product/of South Carolina
    Average 84 stars, based on 28 article reviews
    Price from $9.99 to $1999.99
    pegfp n1 - by Bioz Stars, 2020-04
    84/100 stars
      Buy from Supplier

    92
    Addgene inc plasmids pegfp n1
    Western blot ( IB ) analysis of heterodimerization of α 2B -AR and M6A or Y12A/S13A mutants A, dimerization of α 2B -ARand M6A. HEK293T cells were transfected with HA-tagged α 2B -AR plus the <t>pEGFP-N1</t> vector (control), GFP-tagged α 2B -AR (WT) or GFP-tagged M6A. IP, immunoprecipitation. B, dimerization of α 2B -AR and Y12A/S13A. HEK293T cells were transfected with HA- α 2B -AR together with the pEGFP-N1 vector (control), GFP-tagged α 2B -AR (WT) or GFP-tagged Y12A/S13A. The cells were solubilized and immu-noprecipitated with anti-GFP antibodies. The immunoprecipitate was sepa-rated by SDS-PAGE, and immunoprecipitated receptors were revealed by Western blotting with anti-HAantibodies ( upper panels). The blots were then stripedand reprobed with anti-GFP antibodies ( lower panels). The data shown in A and B are representative of three independent experiments.
    Plasmids Pegfp N1, supplied by Addgene inc, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/plasmids pegfp n1/product/Addgene inc
    Average 92 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    plasmids pegfp n1 - by Bioz Stars, 2020-04
    92/100 stars
      Buy from Supplier

    83
    Stratagene plasmid pegfp n1
    Western blot ( IB ) analysis of heterodimerization of α 2B -AR and M6A or Y12A/S13A mutants A, dimerization of α 2B -ARand M6A. HEK293T cells were transfected with HA-tagged α 2B -AR plus the <t>pEGFP-N1</t> vector (control), GFP-tagged α 2B -AR (WT) or GFP-tagged M6A. IP, immunoprecipitation. B, dimerization of α 2B -AR and Y12A/S13A. HEK293T cells were transfected with HA- α 2B -AR together with the pEGFP-N1 vector (control), GFP-tagged α 2B -AR (WT) or GFP-tagged Y12A/S13A. The cells were solubilized and immu-noprecipitated with anti-GFP antibodies. The immunoprecipitate was sepa-rated by SDS-PAGE, and immunoprecipitated receptors were revealed by Western blotting with anti-HAantibodies ( upper panels). The blots were then stripedand reprobed with anti-GFP antibodies ( lower panels). The data shown in A and B are representative of three independent experiments.
    Plasmid Pegfp N1, supplied by Stratagene, used in various techniques. Bioz Stars score: 83/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/plasmid pegfp n1/product/Stratagene
    Average 83 stars, based on 25 article reviews
    Price from $9.99 to $1999.99
    plasmid pegfp n1 - by Bioz Stars, 2020-04
    83/100 stars
      Buy from Supplier

    92
    TaKaRa fluorescence protein pegfp n1
    Western blot ( IB ) analysis of heterodimerization of α 2B -AR and M6A or Y12A/S13A mutants A, dimerization of α 2B -ARand M6A. HEK293T cells were transfected with HA-tagged α 2B -AR plus the <t>pEGFP-N1</t> vector (control), GFP-tagged α 2B -AR (WT) or GFP-tagged M6A. IP, immunoprecipitation. B, dimerization of α 2B -AR and Y12A/S13A. HEK293T cells were transfected with HA- α 2B -AR together with the pEGFP-N1 vector (control), GFP-tagged α 2B -AR (WT) or GFP-tagged Y12A/S13A. The cells were solubilized and immu-noprecipitated with anti-GFP antibodies. The immunoprecipitate was sepa-rated by SDS-PAGE, and immunoprecipitated receptors were revealed by Western blotting with anti-HAantibodies ( upper panels). The blots were then stripedand reprobed with anti-GFP antibodies ( lower panels). The data shown in A and B are representative of three independent experiments.
    Fluorescence Protein Pegfp N1, supplied by TaKaRa, used in various techniques. Bioz Stars score: 92/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/fluorescence protein pegfp n1/product/TaKaRa
    Average 92 stars, based on 6 article reviews
    Price from $9.99 to $1999.99
    fluorescence protein pegfp n1 - by Bioz Stars, 2020-04
    92/100 stars
      Buy from Supplier

    85
    Vector Biolabs pegfp n1 vector
    Western blot ( IB ) analysis of heterodimerization of α 2B -AR and M6A or Y12A/S13A mutants A, dimerization of α 2B -ARand M6A. HEK293T cells were transfected with HA-tagged α 2B -AR plus the <t>pEGFP-N1</t> vector (control), GFP-tagged α 2B -AR (WT) or GFP-tagged M6A. IP, immunoprecipitation. B, dimerization of α 2B -AR and Y12A/S13A. HEK293T cells were transfected with HA- α 2B -AR together with the pEGFP-N1 vector (control), GFP-tagged α 2B -AR (WT) or GFP-tagged Y12A/S13A. The cells were solubilized and immu-noprecipitated with anti-GFP antibodies. The immunoprecipitate was sepa-rated by SDS-PAGE, and immunoprecipitated receptors were revealed by Western blotting with anti-HAantibodies ( upper panels). The blots were then stripedand reprobed with anti-GFP antibodies ( lower panels). The data shown in A and B are representative of three independent experiments.
    Pegfp N1 Vector, supplied by Vector Biolabs, used in various techniques. Bioz Stars score: 85/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pegfp n1 vector/product/Vector Biolabs
    Average 85 stars, based on 8 article reviews
    Price from $9.99 to $1999.99
    pegfp n1 vector - by Bioz Stars, 2020-04
    85/100 stars
      Buy from Supplier

    94
    Shanghai Genechem pegfp n1
    Western blot ( IB ) analysis of heterodimerization of α 2B -AR and M6A or Y12A/S13A mutants A, dimerization of α 2B -ARand M6A. HEK293T cells were transfected with HA-tagged α 2B -AR plus the <t>pEGFP-N1</t> vector (control), GFP-tagged α 2B -AR (WT) or GFP-tagged M6A. IP, immunoprecipitation. B, dimerization of α 2B -AR and Y12A/S13A. HEK293T cells were transfected with HA- α 2B -AR together with the pEGFP-N1 vector (control), GFP-tagged α 2B -AR (WT) or GFP-tagged Y12A/S13A. The cells were solubilized and immu-noprecipitated with anti-GFP antibodies. The immunoprecipitate was sepa-rated by SDS-PAGE, and immunoprecipitated receptors were revealed by Western blotting with anti-HAantibodies ( upper panels). The blots were then stripedand reprobed with anti-GFP antibodies ( lower panels). The data shown in A and B are representative of three independent experiments.
    Pegfp N1, supplied by Shanghai Genechem, used in various techniques. Bioz Stars score: 94/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pegfp n1/product/Shanghai Genechem
    Average 94 stars, based on 14 article reviews
    Price from $9.99 to $1999.99
    pegfp n1 - by Bioz Stars, 2020-04
    94/100 stars
      Buy from Supplier

    91
    Beyotime pegfp n1
    Western blot ( IB ) analysis of heterodimerization of α 2B -AR and M6A or Y12A/S13A mutants A, dimerization of α 2B -ARand M6A. HEK293T cells were transfected with HA-tagged α 2B -AR plus the <t>pEGFP-N1</t> vector (control), GFP-tagged α 2B -AR (WT) or GFP-tagged M6A. IP, immunoprecipitation. B, dimerization of α 2B -AR and Y12A/S13A. HEK293T cells were transfected with HA- α 2B -AR together with the pEGFP-N1 vector (control), GFP-tagged α 2B -AR (WT) or GFP-tagged Y12A/S13A. The cells were solubilized and immu-noprecipitated with anti-GFP antibodies. The immunoprecipitate was sepa-rated by SDS-PAGE, and immunoprecipitated receptors were revealed by Western blotting with anti-HAantibodies ( upper panels). The blots were then stripedand reprobed with anti-GFP antibodies ( lower panels). The data shown in A and B are representative of three independent experiments.
    Pegfp N1, supplied by Beyotime, used in various techniques. Bioz Stars score: 91/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pegfp n1/product/Beyotime
    Average 91 stars, based on 18 article reviews
    Price from $9.99 to $1999.99
    pegfp n1 - by Bioz Stars, 2020-04
    91/100 stars
      Buy from Supplier

    Image Search Results


    Identification and validation of PTENβ. ( a ) The potential non-AUG initiation codons in favourable Kozak contexts in the 5′ UTR region of Homo sapiens PTEN mRNA are highlighted in red. The initiation codons of PTENα and PTEN are separately highlighted in yellow or green. ( b ) A pEGFP-N1 plasmid containing PTENα with a C-terminal GFP tag was used for detection of PTENα, PTEN and unknown PTEN isoforms (upper panel); the indicated plasmids were introduced in HEK 293T cells followed by western blotting analysis using GFP antibody. β-tubulin was used as a control (lower panel). ( c ) pEGFP-N1 plasmids containing PTEN or PTENα with a C-terminal GFP tag, in which one of the two potential initiation codons (AUU 594 or UUG 621 ) was mutated to CUC combined with mutation of CUG 513 . ( d ) Mutation of AUU 594 but not UUG 621 eliminates PTENβ expression. Plasmids as indicated in c were introduced into HEK 293T cells separately, followed by immunoblotting with GFP antibody. ( e ) The Kozak context of AUU 594 and disruption of the Kozak context by site directed mutagenesis. ( f ) The Kozak context disruption abolishes PTENβ expression. HEK 293T cells were transfected with indicated constructs in e , followed by immunoblotting with GFP antibody. ( g ) A 12 bp AUU 594 downstream palindromic motif in the 5′ UTR of PTEN is evolutionarily conserved. Phylogenetic analysis of the 5′ UTR of PTEN mRNA in bonobo ( Pan paniscus ), killer whale ( Orcinus orca ) and mouse ( Mus musculus ). The alternative CUG 513 and AUU 594 codons are highlighted in blue boxes, and the 12 bp palindromic sequence is highlighted in a red box. ( h ) Disruption of the palindromic motif abolishes PTENβ expression. Upper panel, site directed mutagnesis was used to disrupt the AUU 594 downstream palindromic motif; lower panel, C-terminal GFP-tagged PTENβ expression plasmids with or without disruption of the palindromic motif were introduced into HEK 293T cells, followed by immunoblotting with GFP antibody.

    Journal: Nature Communications

    Article Title: PTENβ is an alternatively translated isoform of PTEN that regulates rDNA transcription

    doi: 10.1038/ncomms14771

    Figure Lengend Snippet: Identification and validation of PTENβ. ( a ) The potential non-AUG initiation codons in favourable Kozak contexts in the 5′ UTR region of Homo sapiens PTEN mRNA are highlighted in red. The initiation codons of PTENα and PTEN are separately highlighted in yellow or green. ( b ) A pEGFP-N1 plasmid containing PTENα with a C-terminal GFP tag was used for detection of PTENα, PTEN and unknown PTEN isoforms (upper panel); the indicated plasmids were introduced in HEK 293T cells followed by western blotting analysis using GFP antibody. β-tubulin was used as a control (lower panel). ( c ) pEGFP-N1 plasmids containing PTEN or PTENα with a C-terminal GFP tag, in which one of the two potential initiation codons (AUU 594 or UUG 621 ) was mutated to CUC combined with mutation of CUG 513 . ( d ) Mutation of AUU 594 but not UUG 621 eliminates PTENβ expression. Plasmids as indicated in c were introduced into HEK 293T cells separately, followed by immunoblotting with GFP antibody. ( e ) The Kozak context of AUU 594 and disruption of the Kozak context by site directed mutagenesis. ( f ) The Kozak context disruption abolishes PTENβ expression. HEK 293T cells were transfected with indicated constructs in e , followed by immunoblotting with GFP antibody. ( g ) A 12 bp AUU 594 downstream palindromic motif in the 5′ UTR of PTEN is evolutionarily conserved. Phylogenetic analysis of the 5′ UTR of PTEN mRNA in bonobo ( Pan paniscus ), killer whale ( Orcinus orca ) and mouse ( Mus musculus ). The alternative CUG 513 and AUU 594 codons are highlighted in blue boxes, and the 12 bp palindromic sequence is highlighted in a red box. ( h ) Disruption of the palindromic motif abolishes PTENβ expression. Upper panel, site directed mutagnesis was used to disrupt the AUU 594 downstream palindromic motif; lower panel, C-terminal GFP-tagged PTENβ expression plasmids with or without disruption of the palindromic motif were introduced into HEK 293T cells, followed by immunoblotting with GFP antibody.

    Article Snippet: Plasmids and cloning strategies The plasmids pCMV-tag-2b, pSA, pEGFP-N1, pDsRed2-N1 and pFastBac1 were purchased from Addgene. pX330 was purchased from BEIJING IDMO CO., LTD. pcDNA3.1-Cas9 was a gift from Dr Jianzhong Xi in the College of Engineering, Peking University.

    Techniques: Plasmid Preparation, Western Blot, Mutagenesis, Expressing, Transfection, Construct, Sequencing

    (a) HLA-ABC and -DR surface expression in transiently transfected RPTECs. Cells were cotransfected with pEGFP-N1 and either pCMV-agno or pCMV-ICP47 at a 1 : 10 ratio. pEGFP-N1 alone (mock, ▲) or together with pCMV-agno ( ♦ ) or with pCMV-ICP47 (□) as described above. MFI: mean fluorescence intensity. (b) HLA-ABC and -DR surface expression in transiently transfected UTA-6. UTA-6 cells were cotransfected with pEGFP-N1 and either pCMV-agno or pCMV-ICP47 at a 1 : 10 ratio. pEGFP-N1 alone (mock, ▲) or together with pCMV-agno ( ♦ ) or with pCMV-ICP47 (□) as described above. MFI: mean fluorescence intensity.

    Journal: Clinical and Developmental Immunology

    Article Title: Comparing Effects of BK Virus Agnoprotein and Herpes Simplex-1 ICP47 on MHC-I and MHC-II Expression

    doi: 10.1155/2013/626823

    Figure Lengend Snippet: (a) HLA-ABC and -DR surface expression in transiently transfected RPTECs. Cells were cotransfected with pEGFP-N1 and either pCMV-agno or pCMV-ICP47 at a 1 : 10 ratio. pEGFP-N1 alone (mock, ▲) or together with pCMV-agno ( ♦ ) or with pCMV-ICP47 (□) as described above. MFI: mean fluorescence intensity. (b) HLA-ABC and -DR surface expression in transiently transfected UTA-6. UTA-6 cells were cotransfected with pEGFP-N1 and either pCMV-agno or pCMV-ICP47 at a 1 : 10 ratio. pEGFP-N1 alone (mock, ▲) or together with pCMV-agno ( ♦ ) or with pCMV-ICP47 (□) as described above. MFI: mean fluorescence intensity.

    Article Snippet: The pCMV-ICP47 and phTRE-ICP47 were generated by replacing the agnoprotein coding sequence with the corresponding sequence encoding ICP47 (a kind gift from Dr. Paul Zajac, University Hospital of Basel, Switzerland). pEGFP-N1 was purchased from BD Biosciences.

    Techniques: Expressing, Transfection, Fluorescence

    HLA-ABC cell surface expression in transfected UTA-6. Cells were cotransfected with pEGFP-N1 and the Tet-off regulated phTRE-agno or pTRE-ICP47 at a 1 : 10 ratio. HLA-ABC expression was analyzed by flow cytometry using labeled antibodies on EGFP-gated UTA-6 cells 48 h after transfection and 24 h after Tet-off induction.

    Journal: Clinical and Developmental Immunology

    Article Title: Comparing Effects of BK Virus Agnoprotein and Herpes Simplex-1 ICP47 on MHC-I and MHC-II Expression

    doi: 10.1155/2013/626823

    Figure Lengend Snippet: HLA-ABC cell surface expression in transfected UTA-6. Cells were cotransfected with pEGFP-N1 and the Tet-off regulated phTRE-agno or pTRE-ICP47 at a 1 : 10 ratio. HLA-ABC expression was analyzed by flow cytometry using labeled antibodies on EGFP-gated UTA-6 cells 48 h after transfection and 24 h after Tet-off induction.

    Article Snippet: The pCMV-ICP47 and phTRE-ICP47 were generated by replacing the agnoprotein coding sequence with the corresponding sequence encoding ICP47 (a kind gift from Dr. Paul Zajac, University Hospital of Basel, Switzerland). pEGFP-N1 was purchased from BD Biosciences.

    Techniques: Expressing, Transfection, Flow Cytometry, Cytometry, Labeling

    (A) HLA-ABC and -DR surface expression in UTA-6 cells transiently transfected with ICP47. HLA-ABC and -DR surface expression was analyzed by flow cytometry of EGFP-gated UTA-6 cells, cotransfected with pEGFP-N1 and pTRE-ICP47, and cultured for 48 hours with different concentration of IFN γ , with (□) or without ( ♦ ) tetracycline. MFI: mean fluorescence intensity. (B) HLA-ABC and -DR surface expression in UTA-6 cells transiently transfected with agnoprotein. HLA-ABC and -DR surface expression was analyzed by flow cytometry of EGFP-gated UTA-6 cells, cotransfected with pEGFP-N1 and phTRE-agno, and cultured for 48 hours with different concentration of IFN γ , with (□) or without ( ♦ ) tetracycline. MFI: mean fluorescence intensity.

    Journal: Clinical and Developmental Immunology

    Article Title: Comparing Effects of BK Virus Agnoprotein and Herpes Simplex-1 ICP47 on MHC-I and MHC-II Expression

    doi: 10.1155/2013/626823

    Figure Lengend Snippet: (A) HLA-ABC and -DR surface expression in UTA-6 cells transiently transfected with ICP47. HLA-ABC and -DR surface expression was analyzed by flow cytometry of EGFP-gated UTA-6 cells, cotransfected with pEGFP-N1 and pTRE-ICP47, and cultured for 48 hours with different concentration of IFN γ , with (□) or without ( ♦ ) tetracycline. MFI: mean fluorescence intensity. (B) HLA-ABC and -DR surface expression in UTA-6 cells transiently transfected with agnoprotein. HLA-ABC and -DR surface expression was analyzed by flow cytometry of EGFP-gated UTA-6 cells, cotransfected with pEGFP-N1 and phTRE-agno, and cultured for 48 hours with different concentration of IFN γ , with (□) or without ( ♦ ) tetracycline. MFI: mean fluorescence intensity.

    Article Snippet: The pCMV-ICP47 and phTRE-ICP47 were generated by replacing the agnoprotein coding sequence with the corresponding sequence encoding ICP47 (a kind gift from Dr. Paul Zajac, University Hospital of Basel, Switzerland). pEGFP-N1 was purchased from BD Biosciences.

    Techniques: Expressing, Transfection, Flow Cytometry, Cytometry, Cell Culture, Concentration Assay, Fluorescence

    Localization of RyR in intracellular membranes of CHO cells. Confocal measurement of green fluorescence in CHO cells transfected with ( A ) pEGFP-N1, ( B ) pcDNA3 (GFP-RyR), and ( C ) pcDNA3 (GFP-RyR-C). Cells expressing GFP alone exhibit a diffuse pattern of fluorescence throughout the cell ( A ), whereas cells expressing the GFP-RyR and GFP-RyR-C fusion proteins show localized fluorescence signal near the perinuclear region of the cells ( B and C ). The panels from left to right show representative sections through the cells at 0.5-μm intervals. The cells have an average thickness of ∼10 μm.

    Journal: The Journal of General Physiology

    Article Title: Caffeine-induced Release of Intracellular Ca2+ from Chinese Hamster Ovary Cells Expressing Skeletal Muscle Ryanodine Receptor

    doi:

    Figure Lengend Snippet: Localization of RyR in intracellular membranes of CHO cells. Confocal measurement of green fluorescence in CHO cells transfected with ( A ) pEGFP-N1, ( B ) pcDNA3 (GFP-RyR), and ( C ) pcDNA3 (GFP-RyR-C). Cells expressing GFP alone exhibit a diffuse pattern of fluorescence throughout the cell ( A ), whereas cells expressing the GFP-RyR and GFP-RyR-C fusion proteins show localized fluorescence signal near the perinuclear region of the cells ( B and C ). The panels from left to right show representative sections through the cells at 0.5-μm intervals. The cells have an average thickness of ∼10 μm.

    Article Snippet: As shown in Fig. , cells transfected with pEGFP-N1 exhibited a diffuse pattern of fluorescence, as expected for a soluble protein (Fig. A ), whereas cells expressing GFP-RyR and GFP-RyR-C fusion proteins exhibited a fluorescence signal only in certain subcellular areas, particularly in the perinuclear region (Fig. , B and C ), indicating that the protein is probably localized to the endoplasmic reticulum (ER) membrane of CHO cells.

    Techniques: Fluorescence, Transfection, Expressing

    Effects of PSMD7 on the activity of mTOR/p70S6K pathway in EC9706 cells and Het‐1A cells. (A) Western blot results of mTOR, p‐mTOR, p70S6K, and p‐p70S6K with GAPDH as internal control. (B and C) The expression of mTOR, p‐mTOR, p70S6K, and p‐p70S6K was analyzed by densitometry using ImageJ software and plotted from three independent experiments. (D) PSMD7 was overexpressed in Het‐1A cells by transfection with the constructed vector pEGFP‐N1‐PSMD7 (OV‐PSMD7) using empty vector as control (Con). The green fluorescence indicated the transfected cells (×100). (E) Western blot results of PSMD7, p‐mTOR, and p‐p70S6K with GAPDH as internal control. (F and G) The expression of PSMD7, p‐mTOR, and p‐p70S6K was analyzed by densitometry using ImageJ software and plotted from three independent experiments. * P

    Journal: FEBS Open Bio

    Article Title: PSMD7 downregulation induces apoptosis and suppresses tumorigenesis of esophageal squamous cell carcinoma via the mTOR/p70S6K pathway

    doi: 10.1002/2211-5463.12394

    Figure Lengend Snippet: Effects of PSMD7 on the activity of mTOR/p70S6K pathway in EC9706 cells and Het‐1A cells. (A) Western blot results of mTOR, p‐mTOR, p70S6K, and p‐p70S6K with GAPDH as internal control. (B and C) The expression of mTOR, p‐mTOR, p70S6K, and p‐p70S6K was analyzed by densitometry using ImageJ software and plotted from three independent experiments. (D) PSMD7 was overexpressed in Het‐1A cells by transfection with the constructed vector pEGFP‐N1‐PSMD7 (OV‐PSMD7) using empty vector as control (Con). The green fluorescence indicated the transfected cells (×100). (E) Western blot results of PSMD7, p‐mTOR, and p‐p70S6K with GAPDH as internal control. (F and G) The expression of PSMD7, p‐mTOR, and p‐p70S6K was analyzed by densitometry using ImageJ software and plotted from three independent experiments. * P

    Article Snippet: Overexpression of PSMD7 in Het‐1A cells The cDNA of PSMD7 was amplified by PCR and inserted into vector pEGFP‐N1 (Clontech, USA) to obtain a recombinated vector pEGFP‐N1‐PSMD7.

    Techniques: Activity Assay, Western Blot, Expressing, Software, Transfection, Construct, Plasmid Preparation, Fluorescence

    Regulation of the viral promoter. (A) HEK-293 cells were transfected with the proviral clone pMtat(−), pEGFP-N1 (normalizing control), and the indicated expression vector in the presence (+) or absence (−) of the Tat-expressing

    Journal:

    Article Title: Role of Cellular RNA Processing Factors in Human Immunodeficiency Virus Type 1 mRNA Metabolism, Replication, and Infectivity ▿

    doi: 10.1128/JVI.01801-08

    Figure Lengend Snippet: Regulation of the viral promoter. (A) HEK-293 cells were transfected with the proviral clone pMtat(−), pEGFP-N1 (normalizing control), and the indicated expression vector in the presence (+) or absence (−) of the Tat-expressing

    Article Snippet: A total of 0.3 μg of plasmid pEGFP-N1 (Clontech) was also added to each transfection mixture as a normalizer for transfection, RNA extraction, and reverse transcription (RT) efficiency.

    Techniques: Transfection, Expressing, Plasmid Preparation

    Downregulation by siRNA. HEK 293 cells were transfected with the proviral clone pNL4-3, pEGFP-N1 (normalizing control), and the indicated siRNA. mRNA was extracted 72 h after transfection and analyzed by qPCR. The six primer sets described in the legend

    Journal:

    Article Title: Role of Cellular RNA Processing Factors in Human Immunodeficiency Virus Type 1 mRNA Metabolism, Replication, and Infectivity ▿

    doi: 10.1128/JVI.01801-08

    Figure Lengend Snippet: Downregulation by siRNA. HEK 293 cells were transfected with the proviral clone pNL4-3, pEGFP-N1 (normalizing control), and the indicated siRNA. mRNA was extracted 72 h after transfection and analyzed by qPCR. The six primer sets described in the legend

    Article Snippet: A total of 0.3 μg of plasmid pEGFP-N1 (Clontech) was also added to each transfection mixture as a normalizer for transfection, RNA extraction, and reverse transcription (RT) efficiency.

    Techniques: Transfection, Real-time Polymerase Chain Reaction

    Overexpression assay. HEK 293 cells were transfected with the proviral clone pNL4-3, pEGFP-N1 (normalizing control), and the indicated expression vector. Each graph summarizes the quantification by qPCR of the indicated mRNA species. (A) Total viral mRNA

    Journal:

    Article Title: Role of Cellular RNA Processing Factors in Human Immunodeficiency Virus Type 1 mRNA Metabolism, Replication, and Infectivity ▿

    doi: 10.1128/JVI.01801-08

    Figure Lengend Snippet: Overexpression assay. HEK 293 cells were transfected with the proviral clone pNL4-3, pEGFP-N1 (normalizing control), and the indicated expression vector. Each graph summarizes the quantification by qPCR of the indicated mRNA species. (A) Total viral mRNA

    Article Snippet: A total of 0.3 μg of plasmid pEGFP-N1 (Clontech) was also added to each transfection mixture as a normalizer for transfection, RNA extraction, and reverse transcription (RT) efficiency.

    Techniques: Over Expression, Transfection, Expressing, Plasmid Preparation, Real-time Polymerase Chain Reaction

    Inhibition of transcription by actinomycin D. HEK 293 cells were transfected with the proviral clone pNL4-3, pEGFP-N1 (normalizing control), and the indicated expression vector or the control plasmid pLuc. Sixty hours later, transfection cells were treated

    Journal:

    Article Title: Role of Cellular RNA Processing Factors in Human Immunodeficiency Virus Type 1 mRNA Metabolism, Replication, and Infectivity ▿

    doi: 10.1128/JVI.01801-08

    Figure Lengend Snippet: Inhibition of transcription by actinomycin D. HEK 293 cells were transfected with the proviral clone pNL4-3, pEGFP-N1 (normalizing control), and the indicated expression vector or the control plasmid pLuc. Sixty hours later, transfection cells were treated

    Article Snippet: A total of 0.3 μg of plasmid pEGFP-N1 (Clontech) was also added to each transfection mixture as a normalizer for transfection, RNA extraction, and reverse transcription (RT) efficiency.

    Techniques: Inhibition, Transfection, Expressing, Plasmid Preparation

    Cellular localization of the viral mRNA. HEK 293 cells were cotransfected with the proviral clone pNL4-3, pEGFP-N1 (normalizing control) and either one of the expression vectors, or treated with siRNA. Nuclear and cytoplasmic RNA fractions were analyzed

    Journal:

    Article Title: Role of Cellular RNA Processing Factors in Human Immunodeficiency Virus Type 1 mRNA Metabolism, Replication, and Infectivity ▿

    doi: 10.1128/JVI.01801-08

    Figure Lengend Snippet: Cellular localization of the viral mRNA. HEK 293 cells were cotransfected with the proviral clone pNL4-3, pEGFP-N1 (normalizing control) and either one of the expression vectors, or treated with siRNA. Nuclear and cytoplasmic RNA fractions were analyzed

    Article Snippet: A total of 0.3 μg of plasmid pEGFP-N1 (Clontech) was also added to each transfection mixture as a normalizer for transfection, RNA extraction, and reverse transcription (RT) efficiency.

    Techniques: Expressing

    Effect of Dex on the AQP5 promoter. EpH4 cells were treated with Dex, 4 h prior ( n = 4) or during transfection ( n = 5) of the luciferase expression pGL3-basic vector including the AQP5 promoter (FF) and the pRL-TK transfection control plasmid (R). Negative controls included the pGL3-basic vector without promoter (cFF) ( n = 2) and the peGFP-N1 vector (GFP), ( n = 1). (a) Levels of AQP5 mRNA normalized to GAPDH are shown for the different transfected EpH4 cells with or without Dex treatment. Bars represent averages and confidence intervals; alpha = 0.05. Significance was tested between samples without Dex and with Dex treatment of the same transfection type. (b) Levels of Firefly enzyme activity normalized to Renilla enzyme activity measured with the luciferase reporter assay for the same transfected cells as in panel (a). Bars represent averages and confidence intervals; alpha = 0.05. Significance was tested between controls (−Dex) and treatments (+Dex), with one or two asterisks denoting P

    Journal: BioMed Research International

    Article Title: Aquaporin 5 Expression in Mouse Mammary Gland Cells Is Not Driven by Promoter Methylation

    doi: 10.1155/2015/460598

    Figure Lengend Snippet: Effect of Dex on the AQP5 promoter. EpH4 cells were treated with Dex, 4 h prior ( n = 4) or during transfection ( n = 5) of the luciferase expression pGL3-basic vector including the AQP5 promoter (FF) and the pRL-TK transfection control plasmid (R). Negative controls included the pGL3-basic vector without promoter (cFF) ( n = 2) and the peGFP-N1 vector (GFP), ( n = 1). (a) Levels of AQP5 mRNA normalized to GAPDH are shown for the different transfected EpH4 cells with or without Dex treatment. Bars represent averages and confidence intervals; alpha = 0.05. Significance was tested between samples without Dex and with Dex treatment of the same transfection type. (b) Levels of Firefly enzyme activity normalized to Renilla enzyme activity measured with the luciferase reporter assay for the same transfected cells as in panel (a). Bars represent averages and confidence intervals; alpha = 0.05. Significance was tested between controls (−Dex) and treatments (+Dex), with one or two asterisks denoting P

    Article Snippet: The same purification procedure was used to obtain high amounts of (promoter-less) pGL3-basic vector (Promega) and the pRL-TK vector (Promega), as well as the peGFP-N1 vector (Clontech) for transfection experiments.

    Techniques: Transfection, Luciferase, Expressing, Plasmid Preparation, Activity Assay, Reporter Assay

    Construction and identification of the DNA vaccine plasmid pEGFP-N1-Catsper1. (A) The amplification of the whole Catsper1 ORF by recombinant PCR. The sequences marked with underline are the restriction enzyme sites of Xho I and Bam H I. (B) Schematic diagram of pEGFP-N1 plasmid. EGFP was used as a report gene, and Kanr/Neor was a kanamycin resistance gene and used for colony screening of E . coli that transformed with plasmid. (C) Agarose gel electrophoresis of the recombinant PCR products. PCR products are, in order, the former half (Segment 1) of Catsper1 ORF with restriction enzyme site (1107 bp), the latter half (Segment 2) of Catsper1 ORF with restriction enzyme site (1106 bp) and the whole Catsper1 ORF with restriction enzyme sites (2079 bp). (D) Agarose gel electrophoresis of restriction enzyme digestion products of recombinant plasmid pEGFP-N1-Catsper1.

    Journal: PLoS ONE

    Article Title: Construction of a Catsper1 DNA Vaccine and Its Antifertility Effect on Male Mice

    doi: 10.1371/journal.pone.0127508

    Figure Lengend Snippet: Construction and identification of the DNA vaccine plasmid pEGFP-N1-Catsper1. (A) The amplification of the whole Catsper1 ORF by recombinant PCR. The sequences marked with underline are the restriction enzyme sites of Xho I and Bam H I. (B) Schematic diagram of pEGFP-N1 plasmid. EGFP was used as a report gene, and Kanr/Neor was a kanamycin resistance gene and used for colony screening of E . coli that transformed with plasmid. (C) Agarose gel electrophoresis of the recombinant PCR products. PCR products are, in order, the former half (Segment 1) of Catsper1 ORF with restriction enzyme site (1107 bp), the latter half (Segment 2) of Catsper1 ORF with restriction enzyme site (1106 bp) and the whole Catsper1 ORF with restriction enzyme sites (2079 bp). (D) Agarose gel electrophoresis of restriction enzyme digestion products of recombinant plasmid pEGFP-N1-Catsper1.

    Article Snippet: One group was immunized with pEGFP-N1-Catsper1 and the other group (control group) with pEGFP-N1 plasmid, both administered the same dosage as mentioned above.

    Techniques: Plasmid Preparation, Amplification, Recombinant, Polymerase Chain Reaction, Colony Assay, Transformation Assay, Agarose Gel Electrophoresis

    Efficient gene expression following electric pulse-mediated gene transfer. Plasmid DNA [65 μg of pEGFP-N1 or 15 μg of Pgc-1 αL and 50 μg of plasmid DNA encoding an empty control vector (pCI-neo) or a mutant/deletion

    Journal: American Journal of Physiology. Cell Physiology

    Article Title: Functional interaction of regulatory factors with the Pgc-1? promoter in response to exercise by in vivo imaging

    doi: 10.1152/ajpcell.00104.2008

    Figure Lengend Snippet: Efficient gene expression following electric pulse-mediated gene transfer. Plasmid DNA [65 μg of pEGFP-N1 or 15 μg of Pgc-1 αL and 50 μg of plasmid DNA encoding an empty control vector (pCI-neo) or a mutant/deletion

    Article Snippet: The dominant-negative ATF2ΔN encodes a FLAG-tagged, truncated activating transcription factor 2 (ATF2) with NH2 -terminal deletion from amino acid 1 to 137 ( ). pCI-neo is a mammalian expression vector without coding region (Promega) used as a negative control for the mutant/deletion expression vectors described above. pEGFP-N1 was purchased from Invitrogen.

    Techniques: Expressing, Plasmid Preparation, Pyrolysis Gas Chromatography, Mutagenesis

    In-vitro analysis in IMR-90 cells. (a) MTT cell viability assay of the DNA/polymer complex with different polymers and at different DNA: polymer weight ratios in IMR-90. The amount of DNA was fixed at 1 μg). (b) Initial testing for PEV-L along with Lipofectamine 2000 (Lip) and PVBLG-8 (P0) with varying pEGFP-N1 plasmid and polymer amount. Transfection efficiency was analysed 48 h post transfection with flow cytometry. (c) Fluorescent images of the transfection using Lip and PEV-H. Scale Bars = 0.25 mm.

    Journal: Biomaterials science

    Article Title: Cationic, helical polypeptide-based gene delivery for IMR-90 fibroblasts and human embryonic stem cells

    doi: 10.1039/C3BM00006K

    Figure Lengend Snippet: In-vitro analysis in IMR-90 cells. (a) MTT cell viability assay of the DNA/polymer complex with different polymers and at different DNA: polymer weight ratios in IMR-90. The amount of DNA was fixed at 1 μg). (b) Initial testing for PEV-L along with Lipofectamine 2000 (Lip) and PVBLG-8 (P0) with varying pEGFP-N1 plasmid and polymer amount. Transfection efficiency was analysed 48 h post transfection with flow cytometry. (c) Fluorescent images of the transfection using Lip and PEV-H. Scale Bars = 0.25 mm.

    Article Snippet: OptiMEM and Lipofectamine 2000 were purchased from Invitrogen (Carlsbad, CA, USA). pEGFP-N1 was obtained from Elim Biopharmaceuticals (Hayward, CA, USA).

    Techniques: In Vitro, MTT Assay, Viability Assay, Plasmid Preparation, Transfection, Flow Cytometry, Cytometry

    Localization of VP4 expressed in COS-7 cells. COS-7 cells were transfected with pEGFP-N1 (C) or pEGFP-N1-VP4 (A and B) and fixed at 48 h posttransfection, and the fluorescence was analyzed by confocal microscopy. Projection of all optical sections (A and C) and three optical sections located at the top, middle, and bottom of the transfected cell (B) are shown. Bar = 20 μm.

    Journal: Journal of Virology

    Article Title: Rotavirus Spike Protein VP4 Is Present at the Plasma Membrane and Is Associated with Microtubules in Infected Cells

    doi:

    Figure Lengend Snippet: Localization of VP4 expressed in COS-7 cells. COS-7 cells were transfected with pEGFP-N1 (C) or pEGFP-N1-VP4 (A and B) and fixed at 48 h posttransfection, and the fluorescence was analyzed by confocal microscopy. Projection of all optical sections (A and C) and three optical sections located at the top, middle, and bottom of the transfected cell (B) are shown. Bar = 20 μm.

    Article Snippet: To put gene 4 upstream of EGFP in pEGFP-N1, the stop codon of gene 4 in pcDNA3-VP4 was replaced by the Pin AI site by site-directed mutagenesis (QuikChange; Stratagene).

    Techniques: Transfection, Fluorescence, Confocal Microscopy

    Specific targeting of scFv5T4. A) Histograms demonstrating surface binding of the indicated antibodies, either commercial mAb5T4 or generated scFv5T4 (empty curves), to 5T4 TAA on colorectal cancer cell line HT-29 measured by FACS. The shaded curves show the IgG2b isotype control (top panel) or streptavidin-FITC alone (bottom panel). B) Visualization of commercial mAb5T4, or scFv5T4, targeting of HEK293 cells transfected with empty vector (pCMV6-XL5) or pCMV6-XL5::5T4 by fluorescence microscopy. Representative images taken at 400× magnification. C) Visualization of HEK293 transfected with pEGFP-N1 or pEGFP-N1::5T4 and incubated with scFv5T4::mRFP1. Same field of view photographs were taken under phase contrast, and green and red fluorescent filters at 100× magnification.

    Journal: PLoS ONE

    Article Title: Control of Established Colon Cancer Xenografts Using a Novel Humanized Single Chain Antibody-Streptococcal Superantigen Fusion Protein Targeting the 5T4 Oncofetal Antigen

    doi: 10.1371/journal.pone.0095200

    Figure Lengend Snippet: Specific targeting of scFv5T4. A) Histograms demonstrating surface binding of the indicated antibodies, either commercial mAb5T4 or generated scFv5T4 (empty curves), to 5T4 TAA on colorectal cancer cell line HT-29 measured by FACS. The shaded curves show the IgG2b isotype control (top panel) or streptavidin-FITC alone (bottom panel). B) Visualization of commercial mAb5T4, or scFv5T4, targeting of HEK293 cells transfected with empty vector (pCMV6-XL5) or pCMV6-XL5::5T4 by fluorescence microscopy. Representative images taken at 400× magnification. C) Visualization of HEK293 transfected with pEGFP-N1 or pEGFP-N1::5T4 and incubated with scFv5T4::mRFP1. Same field of view photographs were taken under phase contrast, and green and red fluorescent filters at 100× magnification.

    Article Snippet: Transfection vectors pCMV6-XL5, pCMV6-XL5::5T4 and pEGFP-N1 were purchased from Origene Technologies, and Clonetech Laboratories, respectively.

    Techniques: Binding Assay, Generated, FACS, Transfection, Plasmid Preparation, Fluorescence, Microscopy, Incubation

    ApoA-I Iowa fibrils were degraded via the autophagy-lysosomal pathway. ( a ) HEK293 cells were plated on poly-L-lysine-coated cover glasses and treated with apoA-I Iowa fibrils (1 μM) at 37 °C for 12 h. Cells were washed with fresh DMEM and then incubated in DMEM at 37 °C for 0, 6, 12, or 24 h. ApoA-I Iowa fibrils were visualized by using an anti-apoA-I antibody and an Alexa Fluor 568-conjugated secondary antibody. ( b ) HEK293 cells were treated with apoA-I Iowa fibrils (1 μM) at 37 °C for 12 h. Cells were washed with fresh DMEM and then incubated in DMEM for 6 h, after which whole cell lysates were prepared. The apoA-I contents of the whole cell lysates were analyzed by means of dot blotting. β-Actin was used as a loading control. The graph shows quantification of cellular apoA-I Iowa fibrils. Data are means ± SE of three independent experiments. ( c , d , e ) HEK293 cells were plated and treated with 1 μM apoA-I Iowa fibrils for 12 h. Cells were washed with fresh DMEM and cultured at 37 °C for an additional 6 or 12 h in fresh DMEM in the presence or absence of chloroquine ( c ; 50 μg/mL, 12 h), 3-MA ( d ; 0.75 μg/mL, 12 h), or rapamycin (e; 2.5 μM, 6 h), after which whole cell lysates were prepared. The apoA-I contents of the whole cell lysates were analyzed by using dot blotting. β-Actin was used as a loading control. ( f ) Effects of TFEB overexpression on cellular degradation of apoA-I Iowa fibrils. HEK293 cells were plated, transfected with pEGFP-N1-TFEB or an empty vector, and cultured at 37 °C for 48 h. Cells were treated with 1 μM apoA-I Iowa fibrils for 12 h, washed with fresh DMEM, and incubated for 12 h, after which whole cell lysates were prepared. The apoA-I contents in whole cell lysates were analyzed by dot blotting with an anti-apoA-I antibody. β-Actin was used as a loading control. The graphs show quantification of the degraded fibrils. Data are means ± SE of three independent experiments. * p = 0.0013 ( c ), 0.011 ( d ), 0.017 ( e ), and 0.032 ( f ) versus each drug or TFEB (−) cells.

    Journal: Scientific Reports

    Article Title: Iowa Mutant Apolipoprotein A-I (ApoA-IIowa) Fibrils Target Lysosomes

    doi: 10.1038/srep30391

    Figure Lengend Snippet: ApoA-I Iowa fibrils were degraded via the autophagy-lysosomal pathway. ( a ) HEK293 cells were plated on poly-L-lysine-coated cover glasses and treated with apoA-I Iowa fibrils (1 μM) at 37 °C for 12 h. Cells were washed with fresh DMEM and then incubated in DMEM at 37 °C for 0, 6, 12, or 24 h. ApoA-I Iowa fibrils were visualized by using an anti-apoA-I antibody and an Alexa Fluor 568-conjugated secondary antibody. ( b ) HEK293 cells were treated with apoA-I Iowa fibrils (1 μM) at 37 °C for 12 h. Cells were washed with fresh DMEM and then incubated in DMEM for 6 h, after which whole cell lysates were prepared. The apoA-I contents of the whole cell lysates were analyzed by means of dot blotting. β-Actin was used as a loading control. The graph shows quantification of cellular apoA-I Iowa fibrils. Data are means ± SE of three independent experiments. ( c , d , e ) HEK293 cells were plated and treated with 1 μM apoA-I Iowa fibrils for 12 h. Cells were washed with fresh DMEM and cultured at 37 °C for an additional 6 or 12 h in fresh DMEM in the presence or absence of chloroquine ( c ; 50 μg/mL, 12 h), 3-MA ( d ; 0.75 μg/mL, 12 h), or rapamycin (e; 2.5 μM, 6 h), after which whole cell lysates were prepared. The apoA-I contents of the whole cell lysates were analyzed by using dot blotting. β-Actin was used as a loading control. ( f ) Effects of TFEB overexpression on cellular degradation of apoA-I Iowa fibrils. HEK293 cells were plated, transfected with pEGFP-N1-TFEB or an empty vector, and cultured at 37 °C for 48 h. Cells were treated with 1 μM apoA-I Iowa fibrils for 12 h, washed with fresh DMEM, and incubated for 12 h, after which whole cell lysates were prepared. The apoA-I contents in whole cell lysates were analyzed by dot blotting with an anti-apoA-I antibody. β-Actin was used as a loading control. The graphs show quantification of the degraded fibrils. Data are means ± SE of three independent experiments. * p = 0.0013 ( c ), 0.011 ( d ), 0.017 ( e ), and 0.032 ( f ) versus each drug or TFEB (−) cells.

    Article Snippet: A monoclonal anti-apoA-I antibody (Wt20-7) was produced as previously described . pEGFP-N1-TFEB was a gift from Shawn Ferguson (Addgene plasmid #38119) .

    Techniques: Incubation, Cell Culture, Over Expression, Transfection, Plasmid Preparation

    Laforin is phosphorylated at residue Ser25. A) Cell extracts from HEK293 cells transfected with plasmids pCMVmyc-laforin (WT, S25D and T194I) were analyzed by 2D-electrophoresis and western blotting using anti-myc monoclonal antibodies. Cell extracts were also treated with λ-phosphatase as described in Experimental section. The calculated pI for each spot is indicated. Molecular size standards are indicated in the right. B) Cell extracts from HEK293 cells co-transfected with plasmids pCMVmyclaforin (WT, S25A and S25D) and pEGFP-N1 (expressing GFP), were prepared for immunodetection as described in Experimental section. In this case, lysis buffer contained 0.5% TritonX100 instead of 0.5% nonidet P40. Extracts were centrifuged at 13,000 rpm for 10 min at 4°C to separate soluble from pellet fractions. These fractions were treated with SDS-PAGE sample buffer and analyzed by western blotting using anti-myc and anti-GFP antibodies. C) HEK293 cells co-transfected with plasmids pCMVmyc-laforin (WT, S25A and S25D) were treated or not with 10 μM MG132 (a proteasome inhibitor) for 8 hours. Then, crude extracts were obtained and analyzed by western blotting using anti-myc and anti-tubulin (loading control).

    Journal: Biochemical Journal

    Article Title: Laforin, a dual specificity protein phosphatase involved in Lafora disease, is phosphorylated at Ser25 by AMP-activated protein kinase

    doi: 10.1042/BJ20110150

    Figure Lengend Snippet: Laforin is phosphorylated at residue Ser25. A) Cell extracts from HEK293 cells transfected with plasmids pCMVmyc-laforin (WT, S25D and T194I) were analyzed by 2D-electrophoresis and western blotting using anti-myc monoclonal antibodies. Cell extracts were also treated with λ-phosphatase as described in Experimental section. The calculated pI for each spot is indicated. Molecular size standards are indicated in the right. B) Cell extracts from HEK293 cells co-transfected with plasmids pCMVmyclaforin (WT, S25A and S25D) and pEGFP-N1 (expressing GFP), were prepared for immunodetection as described in Experimental section. In this case, lysis buffer contained 0.5% TritonX100 instead of 0.5% nonidet P40. Extracts were centrifuged at 13,000 rpm for 10 min at 4°C to separate soluble from pellet fractions. These fractions were treated with SDS-PAGE sample buffer and analyzed by western blotting using anti-myc and anti-GFP antibodies. C) HEK293 cells co-transfected with plasmids pCMVmyc-laforin (WT, S25A and S25D) were treated or not with 10 μM MG132 (a proteasome inhibitor) for 8 hours. Then, crude extracts were obtained and analyzed by western blotting using anti-myc and anti-tubulin (loading control).

    Article Snippet: Plasmid pEGFP-N1 was from BD-Biosciences. pEGB2 AMPKα1 wild type, -kinase dead (KD, AMPKα1 D157A), and -constitutively active (CA, AMPKα1 residues 1-312) were kind gifts from Dr. Reuben Shaw (Salk Institute, San Diego, USA) [ ].

    Techniques: Transfection, Two-Dimensional Gel Electrophoresis, Western Blot, Expressing, Immunodetection, Lysis, SDS Page

    Calsenilin-CTF is responsible for the interaction with RhoA: ( A ) schematic diagram depicting the construction of the pEGFP-N1 vector, calsenilin-FL (aa 1–256), -NTF (aa 1–64), and -CTF (aa 65–256); ( B , C ) total lysates from HEK293 and PC12 cells expressing pEGFP-N1 vector alone (VEC), full-length calsenilin (FL), or truncated fragments (NTF or CTF) were immunoprecipitated with an anti-RhoA antibody and then analyzed by Western blotting with anti-calsenilin (1F11, amino acid 25-33; 4E4), anti-p190RhoGAP and anti-RhoA antibodies; and ( D ) the co-localization of calsenilin with RhoA in HEK293 cells expressing VEC, calsenilin-FL, -NTF, or -CTF was determined by double immunofluorescence staining using confocal microscopy. Green, calsenilin; Red, RhoA; Blue, DAPI. Scale bars, 20 μm.

    Journal: International Journal of Molecular Sciences

    Article Title: Calsenilin, a Presenilin Interactor, Regulates RhoA Signaling and Neurite Outgrowth

    doi: 10.3390/ijms19041196

    Figure Lengend Snippet: Calsenilin-CTF is responsible for the interaction with RhoA: ( A ) schematic diagram depicting the construction of the pEGFP-N1 vector, calsenilin-FL (aa 1–256), -NTF (aa 1–64), and -CTF (aa 65–256); ( B , C ) total lysates from HEK293 and PC12 cells expressing pEGFP-N1 vector alone (VEC), full-length calsenilin (FL), or truncated fragments (NTF or CTF) were immunoprecipitated with an anti-RhoA antibody and then analyzed by Western blotting with anti-calsenilin (1F11, amino acid 25-33; 4E4), anti-p190RhoGAP and anti-RhoA antibodies; and ( D ) the co-localization of calsenilin with RhoA in HEK293 cells expressing VEC, calsenilin-FL, -NTF, or -CTF was determined by double immunofluorescence staining using confocal microscopy. Green, calsenilin; Red, RhoA; Blue, DAPI. Scale bars, 20 μm.

    Article Snippet: The calsenilin–EGFP constructs were made by inserting the FL, NTF, or CTF of calsenilin into the pEGFP-N1 vector (BD Biosciences, Mississauga, ON, Canada) after amplifying the sequences from an existing pcDNA3.1/Zeo(+) calsenilin with the following primers (the endonuclease sites are underlined): FL-EGFP, forward 5′-CACTCGAGGCCACCATGCAGCCGGCTAAGGAA-3′ (XhoI) and reverse 5′-GAGAAGCTTGATGACATTCTCAAACTG-3′ (HindIII); NTF-EGFP, forward 5′-CACTCGAGGCCACCATGCAGCCGGCTAAGGAA-3′ (XhoI) and reverse 5′-GAGAAGCTTGTCGCTGCTATCTGAGCC-3′ (HindIII); and CTF-EGFP, forward 5′-CACTCGAGGCCACCATGAGTGAGCTGGAGCTG-3′ (XhoI) and reverse 5′-GAGAAGCTTGATGACATTCTCAAACTG-3′ (HindIII).

    Techniques: Plasmid Preparation, Expressing, Immunoprecipitation, Western Blot, Double Immunofluorescence Staining, Confocal Microscopy

    Transfection efficiencies mediated by RTNs carrying pEGFP plasmid and assessed by flow cytometry. RTNs were formed using pEGFP-N1 plasmid DNA. Aliquots of nanocomplexes nebulised through the NGI (as in Figure 2A and 3 ) were used to perform transfections. After 48 h incubation, cells were harvested and analysed by flow cytometry to determine the level of expression of enhanced green fluorescent protein (EGFP) in the samples collected from each NGI stage. A representative experiment carried out in normal human bronchial epithelial cell-line 16HBE14o- is shown (cumulative experiments are summarised in Table 2 ). The dot-plot profile of untransfected cells (ctrl) represents the population selected as alive whereas the 1st panel of histogram profile shows where the markers to discriminate the EGFP positive from negative population were set. On each panel the percentage of EGFP negative (left) and EGFP positive (right) cells is indicated. PN = represent cells transfected with pre-nebulisation RTN suspension; LO = cells treated with leftover nanocomplexes in the nebuliser chamber after nebulisation, Thr = throat, and S1–S8 cells transfected with samples from the different NGI stages, respectively.

    Journal: PLoS ONE

    Article Title: Nebulisation of Receptor-Targeted Nanocomplexes for Gene Delivery to the Airway Epithelium

    doi: 10.1371/journal.pone.0026768

    Figure Lengend Snippet: Transfection efficiencies mediated by RTNs carrying pEGFP plasmid and assessed by flow cytometry. RTNs were formed using pEGFP-N1 plasmid DNA. Aliquots of nanocomplexes nebulised through the NGI (as in Figure 2A and 3 ) were used to perform transfections. After 48 h incubation, cells were harvested and analysed by flow cytometry to determine the level of expression of enhanced green fluorescent protein (EGFP) in the samples collected from each NGI stage. A representative experiment carried out in normal human bronchial epithelial cell-line 16HBE14o- is shown (cumulative experiments are summarised in Table 2 ). The dot-plot profile of untransfected cells (ctrl) represents the population selected as alive whereas the 1st panel of histogram profile shows where the markers to discriminate the EGFP positive from negative population were set. On each panel the percentage of EGFP negative (left) and EGFP positive (right) cells is indicated. PN = represent cells transfected with pre-nebulisation RTN suspension; LO = cells treated with leftover nanocomplexes in the nebuliser chamber after nebulisation, Thr = throat, and S1–S8 cells transfected with samples from the different NGI stages, respectively.

    Article Snippet: Plasmid DNA encoding for enhanced green fluorescent protein (pEGFP-N1) was from Clontech (Clontech-Takara Bio Europe, Saint-Germain-en-Laye, France), whereas plasmid pCILuc consists of pCI (Promega, Southampton, UK) carrying a luciferase gene driven by the CMV promoter-enhancer .

    Techniques: Transfection, Plasmid Preparation, Flow Cytometry, Cytometry, Incubation, Expressing

    HCCR-1 is localized on the mitochondria outer membrane . (A) COS-7 (top and second panels), MCF-7 (third and forth panels) and HEK293/HCCR-1-V5 (bottom panel) cells were cultured on the pre-coated coverslip for overnight. COS-7 and MCF-7 cells were transiently transfected with the pEGFP-N1 vector (GFP) or the vector carrying the human HCCR-1 gene tagged with GFP at its COOH-terminus (HCCR-1/GFP). The cells were incubated with MitoTracker (Red), fixed with 4% paraformaldyhyde. HEK293/HCCR-1-V5 cells were stained with MitoTracker (Red) and fixed with 100% methanol for 1 min at -20°C. Mouse anti-V5 antibody (1:300 dilution) and goat anti-mouse IgG conjugated with HRP (5 μg/ml) were applied to the cells for 1 h at room temperature (HCCR-1-V5). The cells were mounted and examined using a confocal microscope. (B) HCCR-1 is expressed at the mitochondrial membrane in the HEK293 cell line. Subcellular fraction of HEK293/HCCR-1-V5 cell line. HEK293/HCCR-1-V5 cells were fractionated into the nuclei (lane 1), post-mitochondria (lane 2), and mitochondria (lane3) fractions by differential centrifugation. Isolated mitochondria were treated with 0.1 M Na 2 CO 3 and fractionated by centrifugation at 100,000 g into pellets (lane 4) and supernatant (lane 5). Proteins were analyzed by immunoblotting using antibodies to the V5 epitope (top panel), the mitochondrial membrane protein VDAC-1 (middle panel) and the mitochondrial matrix protein SOD-2 (bottom panel). (C) HCCR-1 topology on the mitochondrial outer membrane. Intact mitochondria were incubated for 30 min at room temperature without (lane 1) and with 50 μg/ml proteinase K (lane 2) or with 50 μg/ml proteinase K plus 1% Triton X-100 (lane 3). Each sample was precipitated with TCA and separated by SDS-PAGE and analyzed by immunoblotting with antibodies to V5 (top panel), to the outer membrane protein TOM40 (second panel) and to the inner membrane protein TIM23 (third panel), and to the matrix protein SOD-2 (bottom panel). (D) Schematic diagram shows the HCCR-1 topology at the outer mitochondrial membrane.

    Journal: BMC Cell Biology

    Article Title: HCCR-1, a novel oncogene, encodes a mitochondrial outer membrane protein and suppresses the UVC-induced apoptosis

    doi: 10.1186/1471-2121-8-50

    Figure Lengend Snippet: HCCR-1 is localized on the mitochondria outer membrane . (A) COS-7 (top and second panels), MCF-7 (third and forth panels) and HEK293/HCCR-1-V5 (bottom panel) cells were cultured on the pre-coated coverslip for overnight. COS-7 and MCF-7 cells were transiently transfected with the pEGFP-N1 vector (GFP) or the vector carrying the human HCCR-1 gene tagged with GFP at its COOH-terminus (HCCR-1/GFP). The cells were incubated with MitoTracker (Red), fixed with 4% paraformaldyhyde. HEK293/HCCR-1-V5 cells were stained with MitoTracker (Red) and fixed with 100% methanol for 1 min at -20°C. Mouse anti-V5 antibody (1:300 dilution) and goat anti-mouse IgG conjugated with HRP (5 μg/ml) were applied to the cells for 1 h at room temperature (HCCR-1-V5). The cells were mounted and examined using a confocal microscope. (B) HCCR-1 is expressed at the mitochondrial membrane in the HEK293 cell line. Subcellular fraction of HEK293/HCCR-1-V5 cell line. HEK293/HCCR-1-V5 cells were fractionated into the nuclei (lane 1), post-mitochondria (lane 2), and mitochondria (lane3) fractions by differential centrifugation. Isolated mitochondria were treated with 0.1 M Na 2 CO 3 and fractionated by centrifugation at 100,000 g into pellets (lane 4) and supernatant (lane 5). Proteins were analyzed by immunoblotting using antibodies to the V5 epitope (top panel), the mitochondrial membrane protein VDAC-1 (middle panel) and the mitochondrial matrix protein SOD-2 (bottom panel). (C) HCCR-1 topology on the mitochondrial outer membrane. Intact mitochondria were incubated for 30 min at room temperature without (lane 1) and with 50 μg/ml proteinase K (lane 2) or with 50 μg/ml proteinase K plus 1% Triton X-100 (lane 3). Each sample was precipitated with TCA and separated by SDS-PAGE and analyzed by immunoblotting with antibodies to V5 (top panel), to the outer membrane protein TOM40 (second panel) and to the inner membrane protein TIM23 (third panel), and to the matrix protein SOD-2 (bottom panel). (D) Schematic diagram shows the HCCR-1 topology at the outer mitochondrial membrane.

    Article Snippet: The PCR products were subcloned into the pEGFP-N1 vector (Promega, Madison, WI) at Xho I and Bam HI sites and sequenced.

    Techniques: Cell Culture, Transfection, Plasmid Preparation, Incubation, Staining, Microscopy, Centrifugation, Isolation, SDS Page

    Western blot ( IB ) analysis of heterodimerization of α 2B -AR and M6A or Y12A/S13A mutants A, dimerization of α 2B -ARand M6A. HEK293T cells were transfected with HA-tagged α 2B -AR plus the pEGFP-N1 vector (control), GFP-tagged α 2B -AR (WT) or GFP-tagged M6A. IP, immunoprecipitation. B, dimerization of α 2B -AR and Y12A/S13A. HEK293T cells were transfected with HA- α 2B -AR together with the pEGFP-N1 vector (control), GFP-tagged α 2B -AR (WT) or GFP-tagged Y12A/S13A. The cells were solubilized and immu-noprecipitated with anti-GFP antibodies. The immunoprecipitate was sepa-rated by SDS-PAGE, and immunoprecipitated receptors were revealed by Western blotting with anti-HAantibodies ( upper panels). The blots were then stripedand reprobed with anti-GFP antibodies ( lower panels). The data shown in A and B are representative of three independent experiments.

    Journal: The Journal of biological chemistry

    Article Title: Regulation of Anterograde Transport of α2-Adrenergic Receptors by the N Termini at Multiple Intracellular Compartments *

    doi: 10.1074/jbc.M605734200

    Figure Lengend Snippet: Western blot ( IB ) analysis of heterodimerization of α 2B -AR and M6A or Y12A/S13A mutants A, dimerization of α 2B -ARand M6A. HEK293T cells were transfected with HA-tagged α 2B -AR plus the pEGFP-N1 vector (control), GFP-tagged α 2B -AR (WT) or GFP-tagged M6A. IP, immunoprecipitation. B, dimerization of α 2B -AR and Y12A/S13A. HEK293T cells were transfected with HA- α 2B -AR together with the pEGFP-N1 vector (control), GFP-tagged α 2B -AR (WT) or GFP-tagged Y12A/S13A. The cells were solubilized and immu-noprecipitated with anti-GFP antibodies. The immunoprecipitate was sepa-rated by SDS-PAGE, and immunoprecipitated receptors were revealed by Western blotting with anti-HAantibodies ( upper panels). The blots were then stripedand reprobed with anti-GFP antibodies ( lower panels). The data shown in A and B are representative of three independent experiments.

    Article Snippet: For generation of GFP-tagged α 2B -AR-12 construct in which the N-terminal 12 amino acid residues (Ser-2—Ser-13) were deleted from α 2B -AR, the full-length α 2B -AR-GFP was amplified by PCR (forward primer, 5′- GATCAAGCTTATGGTGCAGGCCACCGCCGCCATCGCGTCG-3′; reverse primer, 5′-GATCGTCGACGCCCAGCCAGTCTGGGTC-3′) in which the truncated α 2B -AR was in-frame with GFP, restricted with HindIII and SalI, and ligated into the pEGFP-N1 vector (Invitrogen).

    Techniques: Western Blot, Transfection, Plasmid Preparation, Immunoprecipitation, SDS Page