pcmv6-ac vector Search Results


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  • 91
    OriGene pcmv6 ac gfp vector
    Effects of LGALS4 protein expression (gal-4) in T24 transfectants The cells were transfected with either the <t>pCMV6-AC-GFP/gal-4</t> (T24/gal-4 cell line) or empty vector (T24/mock cell line) as a control. (A) Ectopic expression of gal-4 in T24 transfectants. Proteins from the whole extracts of T24/mock or T24/gal-4 cells were analyzed by Western analysis for the detection of gal-4 or β-actin (as a loading control). The lane beside the markers was run with the extracts of T24 cells as a contrast. T24/gal-4* was T24 transfectants containing the pIRES-EGFP/gal-4 vector, which showed gal-4 expression (36 kD) only as a contrast. (B) Cell proliferation of T24/mock and T24/gal-4 cells, as determined in cell viability assays using trypan blue exclusion method. Bars represent the mean± SEM of three independent experiments performed in duplicate. ** P
    Pcmv6 Ac Gfp Vector, supplied by OriGene, used in various techniques. Bioz Stars score: 91/100, based on 554 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcmv6 ac gfp vector/product/OriGene
    Average 91 stars, based on 554 article reviews
    Price from $9.99 to $1999.99
    pcmv6 ac gfp vector - by Bioz Stars, 2020-09
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    88
    OriGene pcmv6 ac vector
    SPHK1 promotes the lysosomal degradation of CDH1. (A) SPHK1 decreased the expression of CDH1. Cells were transfected with the indicated concentrations of MYC-SPHK1 or vector and protein lysates were analyzed by immunoblotting with the indicated antibodies. (B) SPHK1 did not affect the level of CDH1 mRNA in HepG2 cells. Cells were transfected with the indicated concentrations of MYC-SPHK1 or vector, and total RNA was isolated. CDH1 mRNA was analyzed by fluorescent quantitative RT-PCR, as indicated in Materials and Methods. (C) SPHK1 inhibits the degradation of CDH1 in HepG2 cells. HepG2 cells stably expressing vector or MYC-SPHK1 were transfected with <t>pCMV6-CDH1</t> for 24 h and then treated with CHX (20 μmol/L) for the indicated times. The cell lysates were detected by western blotting using an anti-CDH1 antibody. (D) SPHK1 did not affect the proteasomal degradation of CDH1. HepG2 cells were transfected with pCMV6-CDH1 for 24 h. Cells were treated with MG132 (10 μmol/L) for 2 h, and then also treated with CHX for the indicated times. Immunoblotting was performed with the indicated antibody. (E) SPHK1 accelerated the lysosomal degradation of CDH1. Cells were transfected with pCMV6-CDH1 for 24 h. HepG2 cells were treated with CQ (100 μmol/L) for 12 h, and then CHX was added for the indicated times. Immunoblotting was performed with the indicated antibody. Data are presented as the mean ± SE (n = 4). NS, nonsignificant; CHX, cycloheximide; CQ, chloroquine.
    Pcmv6 Ac Vector, supplied by OriGene, used in various techniques. Bioz Stars score: 88/100, based on 197 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcmv6 ac vector/product/OriGene
    Average 88 stars, based on 197 article reviews
    Price from $9.99 to $1999.99
    pcmv6 ac vector - by Bioz Stars, 2020-09
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    88
    OriGene pcmv6 ac ddk his vector
    SPHK1 promotes the lysosomal degradation of CDH1. (A) SPHK1 decreased the expression of CDH1. Cells were transfected with the indicated concentrations of MYC-SPHK1 or vector and protein lysates were analyzed by immunoblotting with the indicated antibodies. (B) SPHK1 did not affect the level of CDH1 mRNA in HepG2 cells. Cells were transfected with the indicated concentrations of MYC-SPHK1 or vector, and total RNA was isolated. CDH1 mRNA was analyzed by fluorescent quantitative RT-PCR, as indicated in Materials and Methods. (C) SPHK1 inhibits the degradation of CDH1 in HepG2 cells. HepG2 cells stably expressing vector or MYC-SPHK1 were transfected with <t>pCMV6-CDH1</t> for 24 h and then treated with CHX (20 μmol/L) for the indicated times. The cell lysates were detected by western blotting using an anti-CDH1 antibody. (D) SPHK1 did not affect the proteasomal degradation of CDH1. HepG2 cells were transfected with pCMV6-CDH1 for 24 h. Cells were treated with MG132 (10 μmol/L) for 2 h, and then also treated with CHX for the indicated times. Immunoblotting was performed with the indicated antibody. (E) SPHK1 accelerated the lysosomal degradation of CDH1. Cells were transfected with pCMV6-CDH1 for 24 h. HepG2 cells were treated with CQ (100 μmol/L) for 12 h, and then CHX was added for the indicated times. Immunoblotting was performed with the indicated antibody. Data are presented as the mean ± SE (n = 4). NS, nonsignificant; CHX, cycloheximide; CQ, chloroquine.
    Pcmv6 Ac Ddk His Vector, supplied by OriGene, used in various techniques. Bioz Stars score: 88/100, based on 178 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcmv6 ac ddk his vector/product/OriGene
    Average 88 stars, based on 178 article reviews
    Price from $9.99 to $1999.99
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    93
    OriGene pcmv6 ac gfp vectors
    SPHK1 promotes the lysosomal degradation of CDH1. (A) SPHK1 decreased the expression of CDH1. Cells were transfected with the indicated concentrations of MYC-SPHK1 or vector and protein lysates were analyzed by immunoblotting with the indicated antibodies. (B) SPHK1 did not affect the level of CDH1 mRNA in HepG2 cells. Cells were transfected with the indicated concentrations of MYC-SPHK1 or vector, and total RNA was isolated. CDH1 mRNA was analyzed by fluorescent quantitative RT-PCR, as indicated in Materials and Methods. (C) SPHK1 inhibits the degradation of CDH1 in HepG2 cells. HepG2 cells stably expressing vector or MYC-SPHK1 were transfected with <t>pCMV6-CDH1</t> for 24 h and then treated with CHX (20 μmol/L) for the indicated times. The cell lysates were detected by western blotting using an anti-CDH1 antibody. (D) SPHK1 did not affect the proteasomal degradation of CDH1. HepG2 cells were transfected with pCMV6-CDH1 for 24 h. Cells were treated with MG132 (10 μmol/L) for 2 h, and then also treated with CHX for the indicated times. Immunoblotting was performed with the indicated antibody. (E) SPHK1 accelerated the lysosomal degradation of CDH1. Cells were transfected with pCMV6-CDH1 for 24 h. HepG2 cells were treated with CQ (100 μmol/L) for 12 h, and then CHX was added for the indicated times. Immunoblotting was performed with the indicated antibody. Data are presented as the mean ± SE (n = 4). NS, nonsignificant; CHX, cycloheximide; CQ, chloroquine.
    Pcmv6 Ac Gfp Vectors, supplied by OriGene, used in various techniques. Bioz Stars score: 93/100, based on 24 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcmv6 ac gfp vectors/product/OriGene
    Average 93 stars, based on 24 article reviews
    Price from $9.99 to $1999.99
    pcmv6 ac gfp vectors - by Bioz Stars, 2020-09
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    93
    OriGene pcmv6 ac flag vector
    SPHK1 promotes the lysosomal degradation of CDH1. (A) SPHK1 decreased the expression of CDH1. Cells were transfected with the indicated concentrations of MYC-SPHK1 or vector and protein lysates were analyzed by immunoblotting with the indicated antibodies. (B) SPHK1 did not affect the level of CDH1 mRNA in HepG2 cells. Cells were transfected with the indicated concentrations of MYC-SPHK1 or vector, and total RNA was isolated. CDH1 mRNA was analyzed by fluorescent quantitative RT-PCR, as indicated in Materials and Methods. (C) SPHK1 inhibits the degradation of CDH1 in HepG2 cells. HepG2 cells stably expressing vector or MYC-SPHK1 were transfected with <t>pCMV6-CDH1</t> for 24 h and then treated with CHX (20 μmol/L) for the indicated times. The cell lysates were detected by western blotting using an anti-CDH1 antibody. (D) SPHK1 did not affect the proteasomal degradation of CDH1. HepG2 cells were transfected with pCMV6-CDH1 for 24 h. Cells were treated with MG132 (10 μmol/L) for 2 h, and then also treated with CHX for the indicated times. Immunoblotting was performed with the indicated antibody. (E) SPHK1 accelerated the lysosomal degradation of CDH1. Cells were transfected with pCMV6-CDH1 for 24 h. HepG2 cells were treated with CQ (100 μmol/L) for 12 h, and then CHX was added for the indicated times. Immunoblotting was performed with the indicated antibody. Data are presented as the mean ± SE (n = 4). NS, nonsignificant; CHX, cycloheximide; CQ, chloroquine.
    Pcmv6 Ac Flag Vector, supplied by OriGene, used in various techniques. Bioz Stars score: 93/100, based on 37 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcmv6 ac flag vector/product/OriGene
    Average 93 stars, based on 37 article reviews
    Price from $9.99 to $1999.99
    pcmv6 ac flag vector - by Bioz Stars, 2020-09
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    85
    OriGene mammalian expression vector pcmv6 ac
    SPHK1 promotes the lysosomal degradation of CDH1. (A) SPHK1 decreased the expression of CDH1. Cells were transfected with the indicated concentrations of MYC-SPHK1 or vector and protein lysates were analyzed by immunoblotting with the indicated antibodies. (B) SPHK1 did not affect the level of CDH1 mRNA in HepG2 cells. Cells were transfected with the indicated concentrations of MYC-SPHK1 or vector, and total RNA was isolated. CDH1 mRNA was analyzed by fluorescent quantitative RT-PCR, as indicated in Materials and Methods. (C) SPHK1 inhibits the degradation of CDH1 in HepG2 cells. HepG2 cells stably expressing vector or MYC-SPHK1 were transfected with <t>pCMV6-CDH1</t> for 24 h and then treated with CHX (20 μmol/L) for the indicated times. The cell lysates were detected by western blotting using an anti-CDH1 antibody. (D) SPHK1 did not affect the proteasomal degradation of CDH1. HepG2 cells were transfected with pCMV6-CDH1 for 24 h. Cells were treated with MG132 (10 μmol/L) for 2 h, and then also treated with CHX for the indicated times. Immunoblotting was performed with the indicated antibody. (E) SPHK1 accelerated the lysosomal degradation of CDH1. Cells were transfected with pCMV6-CDH1 for 24 h. HepG2 cells were treated with CQ (100 μmol/L) for 12 h, and then CHX was added for the indicated times. Immunoblotting was performed with the indicated antibody. Data are presented as the mean ± SE (n = 4). NS, nonsignificant; CHX, cycloheximide; CQ, chloroquine.
    Mammalian Expression Vector Pcmv6 Ac, supplied by OriGene, used in various techniques. Bioz Stars score: 85/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mammalian expression vector pcmv6 ac/product/OriGene
    Average 85 stars, based on 18 article reviews
    Price from $9.99 to $1999.99
    mammalian expression vector pcmv6 ac - by Bioz Stars, 2020-09
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    90
    OriGene pcmv6 ac gfp mammalian vector
    SPHK1 promotes the lysosomal degradation of CDH1. (A) SPHK1 decreased the expression of CDH1. Cells were transfected with the indicated concentrations of MYC-SPHK1 or vector and protein lysates were analyzed by immunoblotting with the indicated antibodies. (B) SPHK1 did not affect the level of CDH1 mRNA in HepG2 cells. Cells were transfected with the indicated concentrations of MYC-SPHK1 or vector, and total RNA was isolated. CDH1 mRNA was analyzed by fluorescent quantitative RT-PCR, as indicated in Materials and Methods. (C) SPHK1 inhibits the degradation of CDH1 in HepG2 cells. HepG2 cells stably expressing vector or MYC-SPHK1 were transfected with <t>pCMV6-CDH1</t> for 24 h and then treated with CHX (20 μmol/L) for the indicated times. The cell lysates were detected by western blotting using an anti-CDH1 antibody. (D) SPHK1 did not affect the proteasomal degradation of CDH1. HepG2 cells were transfected with pCMV6-CDH1 for 24 h. Cells were treated with MG132 (10 μmol/L) for 2 h, and then also treated with CHX for the indicated times. Immunoblotting was performed with the indicated antibody. (E) SPHK1 accelerated the lysosomal degradation of CDH1. Cells were transfected with pCMV6-CDH1 for 24 h. HepG2 cells were treated with CQ (100 μmol/L) for 12 h, and then CHX was added for the indicated times. Immunoblotting was performed with the indicated antibody. Data are presented as the mean ± SE (n = 4). NS, nonsignificant; CHX, cycloheximide; CQ, chloroquine.
    Pcmv6 Ac Gfp Mammalian Vector, supplied by OriGene, used in various techniques. Bioz Stars score: 90/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcmv6 ac gfp mammalian vector/product/OriGene
    Average 90 stars, based on 21 article reviews
    Price from $9.99 to $1999.99
    pcmv6 ac gfp mammalian vector - by Bioz Stars, 2020-09
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    90
    Blue Heron Biotech pcmv6 ac gfp mammalian expression vector
    SPHK1 promotes the lysosomal degradation of CDH1. (A) SPHK1 decreased the expression of CDH1. Cells were transfected with the indicated concentrations of MYC-SPHK1 or vector and protein lysates were analyzed by immunoblotting with the indicated antibodies. (B) SPHK1 did not affect the level of CDH1 mRNA in HepG2 cells. Cells were transfected with the indicated concentrations of MYC-SPHK1 or vector, and total RNA was isolated. CDH1 mRNA was analyzed by fluorescent quantitative RT-PCR, as indicated in Materials and Methods. (C) SPHK1 inhibits the degradation of CDH1 in HepG2 cells. HepG2 cells stably expressing vector or MYC-SPHK1 were transfected with <t>pCMV6-CDH1</t> for 24 h and then treated with CHX (20 μmol/L) for the indicated times. The cell lysates were detected by western blotting using an anti-CDH1 antibody. (D) SPHK1 did not affect the proteasomal degradation of CDH1. HepG2 cells were transfected with pCMV6-CDH1 for 24 h. Cells were treated with MG132 (10 μmol/L) for 2 h, and then also treated with CHX for the indicated times. Immunoblotting was performed with the indicated antibody. (E) SPHK1 accelerated the lysosomal degradation of CDH1. Cells were transfected with pCMV6-CDH1 for 24 h. HepG2 cells were treated with CQ (100 μmol/L) for 12 h, and then CHX was added for the indicated times. Immunoblotting was performed with the indicated antibody. Data are presented as the mean ± SE (n = 4). NS, nonsignificant; CHX, cycloheximide; CQ, chloroquine.
    Pcmv6 Ac Gfp Mammalian Expression Vector, supplied by Blue Heron Biotech, used in various techniques. Bioz Stars score: 90/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcmv6 ac gfp mammalian expression vector/product/Blue Heron Biotech
    Average 90 stars, based on 15 article reviews
    Price from $9.99 to $1999.99
    pcmv6 ac gfp mammalian expression vector - by Bioz Stars, 2020-09
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    90
    OriGene pcmv6 ac pgrmc2 gfp vector rg204682
    SPHK1 promotes the lysosomal degradation of CDH1. (A) SPHK1 decreased the expression of CDH1. Cells were transfected with the indicated concentrations of MYC-SPHK1 or vector and protein lysates were analyzed by immunoblotting with the indicated antibodies. (B) SPHK1 did not affect the level of CDH1 mRNA in HepG2 cells. Cells were transfected with the indicated concentrations of MYC-SPHK1 or vector, and total RNA was isolated. CDH1 mRNA was analyzed by fluorescent quantitative RT-PCR, as indicated in Materials and Methods. (C) SPHK1 inhibits the degradation of CDH1 in HepG2 cells. HepG2 cells stably expressing vector or MYC-SPHK1 were transfected with <t>pCMV6-CDH1</t> for 24 h and then treated with CHX (20 μmol/L) for the indicated times. The cell lysates were detected by western blotting using an anti-CDH1 antibody. (D) SPHK1 did not affect the proteasomal degradation of CDH1. HepG2 cells were transfected with pCMV6-CDH1 for 24 h. Cells were treated with MG132 (10 μmol/L) for 2 h, and then also treated with CHX for the indicated times. Immunoblotting was performed with the indicated antibody. (E) SPHK1 accelerated the lysosomal degradation of CDH1. Cells were transfected with pCMV6-CDH1 for 24 h. HepG2 cells were treated with CQ (100 μmol/L) for 12 h, and then CHX was added for the indicated times. Immunoblotting was performed with the indicated antibody. Data are presented as the mean ± SE (n = 4). NS, nonsignificant; CHX, cycloheximide; CQ, chloroquine.
    Pcmv6 Ac Pgrmc2 Gfp Vector Rg204682, supplied by OriGene, used in various techniques. Bioz Stars score: 90/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcmv6 ac pgrmc2 gfp vector rg204682/product/OriGene
    Average 90 stars, based on 26 article reviews
    Price from $9.99 to $1999.99
    pcmv6 ac pgrmc2 gfp vector rg204682 - by Bioz Stars, 2020-09
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    93
    Sequetech expression vector pcmv6 ac
    SPHK1 promotes the lysosomal degradation of CDH1. (A) SPHK1 decreased the expression of CDH1. Cells were transfected with the indicated concentrations of MYC-SPHK1 or vector and protein lysates were analyzed by immunoblotting with the indicated antibodies. (B) SPHK1 did not affect the level of CDH1 mRNA in HepG2 cells. Cells were transfected with the indicated concentrations of MYC-SPHK1 or vector, and total RNA was isolated. CDH1 mRNA was analyzed by fluorescent quantitative RT-PCR, as indicated in Materials and Methods. (C) SPHK1 inhibits the degradation of CDH1 in HepG2 cells. HepG2 cells stably expressing vector or MYC-SPHK1 were transfected with <t>pCMV6-CDH1</t> for 24 h and then treated with CHX (20 μmol/L) for the indicated times. The cell lysates were detected by western blotting using an anti-CDH1 antibody. (D) SPHK1 did not affect the proteasomal degradation of CDH1. HepG2 cells were transfected with pCMV6-CDH1 for 24 h. Cells were treated with MG132 (10 μmol/L) for 2 h, and then also treated with CHX for the indicated times. Immunoblotting was performed with the indicated antibody. (E) SPHK1 accelerated the lysosomal degradation of CDH1. Cells were transfected with pCMV6-CDH1 for 24 h. HepG2 cells were treated with CQ (100 μmol/L) for 12 h, and then CHX was added for the indicated times. Immunoblotting was performed with the indicated antibody. Data are presented as the mean ± SE (n = 4). NS, nonsignificant; CHX, cycloheximide; CQ, chloroquine.
    Expression Vector Pcmv6 Ac, supplied by Sequetech, used in various techniques. Bioz Stars score: 93/100, based on 24 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/expression vector pcmv6 ac/product/Sequetech
    Average 93 stars, based on 24 article reviews
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    expression vector pcmv6 ac - by Bioz Stars, 2020-09
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    92
    OriGene l521f mutant pcmv6 ac gfp vector
    Expression of CLCN5 constructs in human podocyte cell lines. Western blot (WB) showed a single band consistent with the short 83-kDa CLCN5 proteoform. Neph1 expression, which was used a positive control, was confirmed using an anti-Neph1 antibody (Ab; a). Endogenous expression of CLCN5 was also determined by immunofluorescence in cultured human podocytes (b). The upper panel shows human podocytes transfected with green fluorescent protein <t>(GFP)–tagged</t> wild-type (WT) CLCN5 protein demonstrated predominantly cell surface distribution and co-localization with cell surface marker ZO-1. The lower panel shows cultured human podocytes transfected with GFP-tagged <t>L521F</t> mutant CLCN5 protein, which demonstrated predominantly intracellular distribution (c). DAPI, 4′,6-diamidino-2-phenylindole.
    L521f Mutant Pcmv6 Ac Gfp Vector, supplied by OriGene, used in various techniques. Bioz Stars score: 92/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/l521f mutant pcmv6 ac gfp vector/product/OriGene
    Average 92 stars, based on 10 article reviews
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    l521f mutant pcmv6 ac gfp vector - by Bioz Stars, 2020-09
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    Image Search Results


    Effects of LGALS4 protein expression (gal-4) in T24 transfectants The cells were transfected with either the pCMV6-AC-GFP/gal-4 (T24/gal-4 cell line) or empty vector (T24/mock cell line) as a control. (A) Ectopic expression of gal-4 in T24 transfectants. Proteins from the whole extracts of T24/mock or T24/gal-4 cells were analyzed by Western analysis for the detection of gal-4 or β-actin (as a loading control). The lane beside the markers was run with the extracts of T24 cells as a contrast. T24/gal-4* was T24 transfectants containing the pIRES-EGFP/gal-4 vector, which showed gal-4 expression (36 kD) only as a contrast. (B) Cell proliferation of T24/mock and T24/gal-4 cells, as determined in cell viability assays using trypan blue exclusion method. Bars represent the mean± SEM of three independent experiments performed in duplicate. ** P

    Journal: Oncotarget

    Article Title: Promoter hypermethylation of LGALS4 correlates with poor prognosis in patients with urothelial carcinoma

    doi: 10.18632/oncotarget.15865

    Figure Lengend Snippet: Effects of LGALS4 protein expression (gal-4) in T24 transfectants The cells were transfected with either the pCMV6-AC-GFP/gal-4 (T24/gal-4 cell line) or empty vector (T24/mock cell line) as a control. (A) Ectopic expression of gal-4 in T24 transfectants. Proteins from the whole extracts of T24/mock or T24/gal-4 cells were analyzed by Western analysis for the detection of gal-4 or β-actin (as a loading control). The lane beside the markers was run with the extracts of T24 cells as a contrast. T24/gal-4* was T24 transfectants containing the pIRES-EGFP/gal-4 vector, which showed gal-4 expression (36 kD) only as a contrast. (B) Cell proliferation of T24/mock and T24/gal-4 cells, as determined in cell viability assays using trypan blue exclusion method. Bars represent the mean± SEM of three independent experiments performed in duplicate. ** P

    Article Snippet: The PCR product was digested and subcloned into the pCMV6-AC-GFP vector (Origene, Rockville, MD, USA) under the control of a CMV promoter.

    Techniques: Expressing, Transfection, Plasmid Preparation, Western Blot

    SPHK1 promotes the lysosomal degradation of CDH1. (A) SPHK1 decreased the expression of CDH1. Cells were transfected with the indicated concentrations of MYC-SPHK1 or vector and protein lysates were analyzed by immunoblotting with the indicated antibodies. (B) SPHK1 did not affect the level of CDH1 mRNA in HepG2 cells. Cells were transfected with the indicated concentrations of MYC-SPHK1 or vector, and total RNA was isolated. CDH1 mRNA was analyzed by fluorescent quantitative RT-PCR, as indicated in Materials and Methods. (C) SPHK1 inhibits the degradation of CDH1 in HepG2 cells. HepG2 cells stably expressing vector or MYC-SPHK1 were transfected with pCMV6-CDH1 for 24 h and then treated with CHX (20 μmol/L) for the indicated times. The cell lysates were detected by western blotting using an anti-CDH1 antibody. (D) SPHK1 did not affect the proteasomal degradation of CDH1. HepG2 cells were transfected with pCMV6-CDH1 for 24 h. Cells were treated with MG132 (10 μmol/L) for 2 h, and then also treated with CHX for the indicated times. Immunoblotting was performed with the indicated antibody. (E) SPHK1 accelerated the lysosomal degradation of CDH1. Cells were transfected with pCMV6-CDH1 for 24 h. HepG2 cells were treated with CQ (100 μmol/L) for 12 h, and then CHX was added for the indicated times. Immunoblotting was performed with the indicated antibody. Data are presented as the mean ± SE (n = 4). NS, nonsignificant; CHX, cycloheximide; CQ, chloroquine.

    Journal: Autophagy

    Article Title: SPHK1 (sphingosine kinase 1) induces epithelial-mesenchymal transition by promoting the autophagy-linked lysosomal degradation of CDH1/E-cadherin in hepatoma cells

    doi: 10.1080/15548627.2017.1291479

    Figure Lengend Snippet: SPHK1 promotes the lysosomal degradation of CDH1. (A) SPHK1 decreased the expression of CDH1. Cells were transfected with the indicated concentrations of MYC-SPHK1 or vector and protein lysates were analyzed by immunoblotting with the indicated antibodies. (B) SPHK1 did not affect the level of CDH1 mRNA in HepG2 cells. Cells were transfected with the indicated concentrations of MYC-SPHK1 or vector, and total RNA was isolated. CDH1 mRNA was analyzed by fluorescent quantitative RT-PCR, as indicated in Materials and Methods. (C) SPHK1 inhibits the degradation of CDH1 in HepG2 cells. HepG2 cells stably expressing vector or MYC-SPHK1 were transfected with pCMV6-CDH1 for 24 h and then treated with CHX (20 μmol/L) for the indicated times. The cell lysates were detected by western blotting using an anti-CDH1 antibody. (D) SPHK1 did not affect the proteasomal degradation of CDH1. HepG2 cells were transfected with pCMV6-CDH1 for 24 h. Cells were treated with MG132 (10 μmol/L) for 2 h, and then also treated with CHX for the indicated times. Immunoblotting was performed with the indicated antibody. (E) SPHK1 accelerated the lysosomal degradation of CDH1. Cells were transfected with pCMV6-CDH1 for 24 h. HepG2 cells were treated with CQ (100 μmol/L) for 12 h, and then CHX was added for the indicated times. Immunoblotting was performed with the indicated antibody. Data are presented as the mean ± SE (n = 4). NS, nonsignificant; CHX, cycloheximide; CQ, chloroquine.

    Article Snippet: HA-tagged BECN1 was constructed in the pCMV6-AC-HA vector (PS100004, Origene) by standard subcloning.

    Techniques: Expressing, Transfection, Plasmid Preparation, Isolation, Quantitative RT-PCR, Stable Transfection, Western Blot

    SPHK1 induces the EMT by stimulating BECN1 in HepG2 cells. (A, B) Silencing BECN1 blocked cell migration and invasion in SPHK1-overexpressing cells. HepG2 cells stably expressing vector or MYC-SPHK1 were transfected with control-siRNA or si- BECN1 and plated in the upper chamber of the filters for 24 h. Then cells migrating to the underside of the transwell insert were counted. (C) SPHK1 overexpression regulated the expression of EMT-related markers through stimulating BECN1. HepG2 cells stably expressing vector or MYC-SPHK1 were transfected with control-siRNA or si- BECN1 for 24 h and then harvested for cell lysate extraction. The level of epithelial or mesenchymal markers was detected by western blot analysis. (D) Silencing BECN1 blocked the degradation of CDH1 in SPHK1-overexpressing cells. HepG2 cells stably expressing MYC-SPHK1 were cotransfected with pCMV6-CDH1 and si- BECN1 (or control-siRNA) for 24 h and then treated with CHX (20 μmol/L) for the indicated times. The cell lysates were analyzed by western blotting using an anti-CDH1 antibody. (E) Schematic diagram of the mechanism of SPHK1-mediated autophagy and lysosomal CDH1 degradation that stimulates the EMT in HepG2 cells. Con-siRNA, control-siRNA.

    Journal: Autophagy

    Article Title: SPHK1 (sphingosine kinase 1) induces epithelial-mesenchymal transition by promoting the autophagy-linked lysosomal degradation of CDH1/E-cadherin in hepatoma cells

    doi: 10.1080/15548627.2017.1291479

    Figure Lengend Snippet: SPHK1 induces the EMT by stimulating BECN1 in HepG2 cells. (A, B) Silencing BECN1 blocked cell migration and invasion in SPHK1-overexpressing cells. HepG2 cells stably expressing vector or MYC-SPHK1 were transfected with control-siRNA or si- BECN1 and plated in the upper chamber of the filters for 24 h. Then cells migrating to the underside of the transwell insert were counted. (C) SPHK1 overexpression regulated the expression of EMT-related markers through stimulating BECN1. HepG2 cells stably expressing vector or MYC-SPHK1 were transfected with control-siRNA or si- BECN1 for 24 h and then harvested for cell lysate extraction. The level of epithelial or mesenchymal markers was detected by western blot analysis. (D) Silencing BECN1 blocked the degradation of CDH1 in SPHK1-overexpressing cells. HepG2 cells stably expressing MYC-SPHK1 were cotransfected with pCMV6-CDH1 and si- BECN1 (or control-siRNA) for 24 h and then treated with CHX (20 μmol/L) for the indicated times. The cell lysates were analyzed by western blotting using an anti-CDH1 antibody. (E) Schematic diagram of the mechanism of SPHK1-mediated autophagy and lysosomal CDH1 degradation that stimulates the EMT in HepG2 cells. Con-siRNA, control-siRNA.

    Article Snippet: HA-tagged BECN1 was constructed in the pCMV6-AC-HA vector (PS100004, Origene) by standard subcloning.

    Techniques: Migration, Stable Transfection, Expressing, Plasmid Preparation, Transfection, Over Expression, Western Blot

    Alpha-galactosidase activity resulting from 1-deoxy-galactonojirimycin administration in transfected cells. Comparison between different cell hosts and transfection methods. COS7 or HEK293 cells were co-transfected with pCMV6-AC harboring L300F-AGAL gene and with pMIR vector harboring the luciferase reporter gene. Transfections were performed with Lipofectamine® ltx, Fugene® HD or CalPhos™ Mammalian Transfection Kit, either using cell in adhesion (A) or in suspension (S). Cells were than cultivated with (+) or without (−) 0.02 mM DGJ. After cell lysis AGAL activity and luciferase activity were measured. Four independent experiments were performed in the presence of DGJ, two without DGJ. Alpha galactosidase activity (expressed as mut_activity / WT_activity − DGJ × 100) was normalized by protein concentration in panel A and by luciferase activity in panel B.

    Journal: Biochimica et Biophysica Acta

    Article Title: A thermodynamic assay to test pharmacological chaperones for Fabry disease

    doi: 10.1016/j.bbagen.2013.12.018

    Figure Lengend Snippet: Alpha-galactosidase activity resulting from 1-deoxy-galactonojirimycin administration in transfected cells. Comparison between different cell hosts and transfection methods. COS7 or HEK293 cells were co-transfected with pCMV6-AC harboring L300F-AGAL gene and with pMIR vector harboring the luciferase reporter gene. Transfections were performed with Lipofectamine® ltx, Fugene® HD or CalPhos™ Mammalian Transfection Kit, either using cell in adhesion (A) or in suspension (S). Cells were than cultivated with (+) or without (−) 0.02 mM DGJ. After cell lysis AGAL activity and luciferase activity were measured. Four independent experiments were performed in the presence of DGJ, two without DGJ. Alpha galactosidase activity (expressed as mut_activity / WT_activity − DGJ × 100) was normalized by protein concentration in panel A and by luciferase activity in panel B.

    Article Snippet: 4.1 Cell cultures The clone SC319065, which contains the full length cDNA for wild type human AGAL inserted into the expression vector pCMV6-AC, was purchased from Origene (Rockville, MD, USA).

    Techniques: Activity Assay, Transfection, Plasmid Preparation, Luciferase, Lysis, Protein Concentration

    Expression of CLCN5 constructs in human podocyte cell lines. Western blot (WB) showed a single band consistent with the short 83-kDa CLCN5 proteoform. Neph1 expression, which was used a positive control, was confirmed using an anti-Neph1 antibody (Ab; a). Endogenous expression of CLCN5 was also determined by immunofluorescence in cultured human podocytes (b). The upper panel shows human podocytes transfected with green fluorescent protein (GFP)–tagged wild-type (WT) CLCN5 protein demonstrated predominantly cell surface distribution and co-localization with cell surface marker ZO-1. The lower panel shows cultured human podocytes transfected with GFP-tagged L521F mutant CLCN5 protein, which demonstrated predominantly intracellular distribution (c). DAPI, 4′,6-diamidino-2-phenylindole.

    Journal: Kidney International Reports

    Article Title: A Novel CLCN5 Mutation Associated With Focal Segmental Glomerulosclerosis and Podocyte Injury

    doi: 10.1016/j.ekir.2018.06.003

    Figure Lengend Snippet: Expression of CLCN5 constructs in human podocyte cell lines. Western blot (WB) showed a single band consistent with the short 83-kDa CLCN5 proteoform. Neph1 expression, which was used a positive control, was confirmed using an anti-Neph1 antibody (Ab; a). Endogenous expression of CLCN5 was also determined by immunofluorescence in cultured human podocytes (b). The upper panel shows human podocytes transfected with green fluorescent protein (GFP)–tagged wild-type (WT) CLCN5 protein demonstrated predominantly cell surface distribution and co-localization with cell surface marker ZO-1. The lower panel shows cultured human podocytes transfected with GFP-tagged L521F mutant CLCN5 protein, which demonstrated predominantly intracellular distribution (c). DAPI, 4′,6-diamidino-2-phenylindole.

    Article Snippet: The pCMV6-AC-GFP vector carrying the wild-type CLCN5 gene (WT_CLCN5) and L521F mutant pCMV6-AC-GFP vector (L521F_CLCN5) were purchased from Origene (Rockville, MD).

    Techniques: Expressing, Construct, Western Blot, Positive Control, Immunofluorescence, Cell Culture, Transfection, Marker, Mutagenesis