Journal: Digestive diseases and sciences

Article Title: Modulation of function of sodium-dependent vitamin C transporter 1 (SVCT1) by Rab8a in intestinal epithelial cells: Studies utilizing Caco-2 cells and Rab8a knockout mice

doi: 10.1007/s10620-012-2388-9

Figure Lengend Snippet: Primer sequences to clone hSVCT1 and Rab8a and real-time PCR primers

Article Snippet: Cell surface biotinylation Control and Rab8a siRNAs (Santa Cruz) were transiently transfected into Caco-2 cells (90% confluent).

Techniques: Real-time Polymerase Chain Reaction

Journal: Digestive diseases and sciences

Article Title: Modulation of function of sodium-dependent vitamin C transporter 1 (SVCT1) by Rab8a in intestinal epithelial cells: Studies utilizing Caco-2 cells and Rab8a knockout mice

doi: 10.1007/s10620-012-2388-9

Figure Lengend Snippet: Caco-2, HT-29, NCM460 and HuTU-80 cells were transiently co-transfected with hSVCT1-YEP and DsRed-Rab8a constructs. Data are from n > 6-10 transfected cells. Scale bar is 10 μm

Article Snippet: Cell surface biotinylation Control and Rab8a siRNAs (Santa Cruz) were transiently transfected into Caco-2 cells (90% confluent).

Techniques: Transfection, Construct

Journal: Digestive diseases and sciences

Article Title: Modulation of function of sodium-dependent vitamin C transporter 1 (SVCT1) by Rab8a in intestinal epithelial cells: Studies utilizing Caco-2 cells and Rab8a knockout mice

doi: 10.1007/s10620-012-2388-9

Figure Lengend Snippet: A) 14C-AA uptake (32 μM) was performed on Rab8a siRNA treated hSVCT1 expressing Caco-2 cells. Data are mean ± SE of at least three separate experiments performed on different batches of cells on separate occasions. *p < 0.02. B) Real-time PCR was performed using gene specific primers for Rab8a and β-actin from total RNA isolated from Rab8a siRNA and control siRNA (scrambled) treated Caco-2 cells. Data are mean ± SE of at least three independent experiments. *p < 0.01. C, Top, Western blot analysis was performed on cell extract (60 μg) isolated from control (left) and Rab8a siRNAs treated Caco-2 cells (right). Blots were incubated with rabbit polyclonal anti-human Rab8a specific antibodies (top) along with β-actin antibodies (bottom). Bottom, densitometric quantification of the immunoreactive bands. Data are mean ± SE of at least three separate sample preparations. *p < 0.01. D) Carrier-mediated 14C-AA uptake (32 μM) by Rab8a KO mice jejunal portion was performed as described in “Materials and Methods”. Values are mean ± SE of at least three separate uptake determinations from multiple sets of mice. *p < 0.01. E) Real-time PCR was performed on total RNA isolated from Rab8a KO and wild-type (litter-mate) mice jejunal mucosa using mouse Rab8a and β-actin gene-specific primers. Data are mean ± SE of at least three separate samples from three different mice. *p < 0.01. F, Top) Western blot analysis was performed on Rab8a KO (right) and wild-type litter-mate (left) mouse jejunum mucosal (60 μg) proteins. Blots were incubated with rabbit polyclonal anti-mouse Rab8a antibodies along with β-actin antibodies. Bottom, densitometric values. Data are mean ± SE of at least three sets of samples from three different mice.

Article Snippet: Cell surface biotinylation Control and Rab8a siRNAs (Santa Cruz) were transiently transfected into Caco-2 cells (90% confluent).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Isolation, Control, Western Blot, Incubation

Journal: Digestive diseases and sciences

Article Title: Modulation of function of sodium-dependent vitamin C transporter 1 (SVCT1) by Rab8a in intestinal epithelial cells: Studies utilizing Caco-2 cells and Rab8a knockout mice

doi: 10.1007/s10620-012-2388-9

Figure Lengend Snippet: A) Real-time PCR was performed using primers for hSVCT1, hSVCT2 and β-actin on total RNA isolated from control (scrambled) and Rab8a siRNAs treated Caco-2 cells. Data are mean ± SE of multiple experiments performed on different batches of cells. B, Top, western blot analysis was performed on cell extract (60 μg) isolated from control and Rab8a siRNAs treated Caco-2 cells. Blots were incubated with rabbit anti-human hSVCT1 specific antibodies along with β-actin antibodies. Bottom, densitometric values. Data are mean ± SE of multiple experiments performed on separately isolated samples.*p < 0.01. C) Real-time PCR was performed on total RNA isolated from Rab8a KO and wild-type litter-mate mice jejunal mucosa using mouse SVCT1, SVCT2 and β-actin primers. Data are mean ± SE of at least three separate samples from three different mice. D, Top, western blot analysis was performed on Rab8a KO and wild-type (litter-mate) mouse jejunum mucosal (60 μg) proteins. Blots were incubated with rabbit anti-mouse SVCT1 antibodies along with β-actin antibodies. Bottom, densitometric values. Data are mean ± SE of at least three separate samples from three mice. *p < 0.02.

Article Snippet: Cell surface biotinylation Control and Rab8a siRNAs (Santa Cruz) were transiently transfected into Caco-2 cells (90% confluent).

Techniques: Real-time Polymerase Chain Reaction, Isolation, Control, Western Blot, Incubation

Journal: Digestive diseases and sciences

Article Title: Modulation of function of sodium-dependent vitamin C transporter 1 (SVCT1) by Rab8a in intestinal epithelial cells: Studies utilizing Caco-2 cells and Rab8a knockout mice

doi: 10.1007/s10620-012-2388-9

Figure Lengend Snippet: Caco-2 cells were transiently transfected with Rab8a siRNA or control siRNA. Forty eight hours after transfection cells were processed for biotinylation. Equal amount of protein was loaded onto pre-made 4-12% mini-gel and western blot was performed using anti-hSVCT1 antibody. The level of cell surface expression was normalized relative to the total amount of cellular hSVCT1 protein. Densitometric values are from mean ± SE of four independent experiments. Inset shows representative western blot images. *p < 0.01.

Article Snippet: Cell surface biotinylation Control and Rab8a siRNAs (Santa Cruz) were transiently transfected into Caco-2 cells (90% confluent).

Techniques: Transfection, Control, Western Blot, Expressing

Journal: Digestive diseases and sciences

Article Title: Modulation of function of sodium-dependent vitamin C transporter 1 (SVCT1) by Rab8a in intestinal epithelial cells: Studies utilizing Caco-2 cells and Rab8a knockout mice

doi: 10.1007/s10620-012-2388-9

Figure Lengend Snippet: Hu-Tu-80 cells were co-transfected with hSVCT1-YEP, LAMP1-RFP, control or Rab8a siRNAs. Live cell confocal imaging (laterl sections-xy) was performed after 48 h of transfection. Data are from n > 6-10 transfected cells and representative images were shown. Scale bar is 10 μm.

Article Snippet: Cell surface biotinylation Control and Rab8a siRNAs (Santa Cruz) were transiently transfected into Caco-2 cells (90% confluent).

Techniques: Transfection, Control, Imaging

Journal: Digestive diseases and sciences

Article Title: Modulation of function of sodium-dependent vitamin C transporter 1 (SVCT1) by Rab8a in intestinal epithelial cells: Studies utilizing Caco-2 cells and Rab8a knockout mice

doi: 10.1007/s10620-012-2388-9

Figure Lengend Snippet: Primer sequences to clone hSVCT1 and Rab8a and real-time PCR primers

Article Snippet: For uptake, mRNA, and western blot analysis, Caco-2 cells were grown on 12 well plats (Corning Inc., NY) and Rab8a siRNA [pool of three different siRNA duplexes (duplex-1, sense: GAACUGGAUUCGCAACAUUtt, antisense: AAUGUUGCGAAUCCAGUUCtt; duplex-2, sense: GAACAAGUGUGAUGUGAAUtt, antisense: AUUCACAUCACACUUGUUCtt; duplex-3, sense: CCAGAAUGCAAUUGAGAAAtt, antisense: UUUCUCAAUUGCAUUCUGGtt; specific for human Rab8a (Santa Cruz, CA)] was transfected at 80% confluence.

Techniques: Real-time Polymerase Chain Reaction

Journal: Digestive diseases and sciences

Article Title: Modulation of function of sodium-dependent vitamin C transporter 1 (SVCT1) by Rab8a in intestinal epithelial cells: Studies utilizing Caco-2 cells and Rab8a knockout mice

doi: 10.1007/s10620-012-2388-9

Figure Lengend Snippet: Caco-2, HT-29, NCM460 and HuTU-80 cells were transiently co-transfected with hSVCT1-YEP and DsRed-Rab8a constructs. Data are from n > 6-10 transfected cells. Scale bar is 10 μm

Article Snippet: For uptake, mRNA, and western blot analysis, Caco-2 cells were grown on 12 well plats (Corning Inc., NY) and Rab8a siRNA [pool of three different siRNA duplexes (duplex-1, sense: GAACUGGAUUCGCAACAUUtt, antisense: AAUGUUGCGAAUCCAGUUCtt; duplex-2, sense: GAACAAGUGUGAUGUGAAUtt, antisense: AUUCACAUCACACUUGUUCtt; duplex-3, sense: CCAGAAUGCAAUUGAGAAAtt, antisense: UUUCUCAAUUGCAUUCUGGtt; specific for human Rab8a (Santa Cruz, CA)] was transfected at 80% confluence.

Techniques: Transfection, Construct

Journal: Digestive diseases and sciences

Article Title: Modulation of function of sodium-dependent vitamin C transporter 1 (SVCT1) by Rab8a in intestinal epithelial cells: Studies utilizing Caco-2 cells and Rab8a knockout mice

doi: 10.1007/s10620-012-2388-9

Figure Lengend Snippet: A) 14C-AA uptake (32 μM) was performed on Rab8a siRNA treated hSVCT1 expressing Caco-2 cells. Data are mean ± SE of at least three separate experiments performed on different batches of cells on separate occasions. *p < 0.02. B) Real-time PCR was performed using gene specific primers for Rab8a and β-actin from total RNA isolated from Rab8a siRNA and control siRNA (scrambled) treated Caco-2 cells. Data are mean ± SE of at least three independent experiments. *p < 0.01. C, Top, Western blot analysis was performed on cell extract (60 μg) isolated from control (left) and Rab8a siRNAs treated Caco-2 cells (right). Blots were incubated with rabbit polyclonal anti-human Rab8a specific antibodies (top) along with β-actin antibodies (bottom). Bottom, densitometric quantification of the immunoreactive bands. Data are mean ± SE of at least three separate sample preparations. *p < 0.01. D) Carrier-mediated 14C-AA uptake (32 μM) by Rab8a KO mice jejunal portion was performed as described in “Materials and Methods”. Values are mean ± SE of at least three separate uptake determinations from multiple sets of mice. *p < 0.01. E) Real-time PCR was performed on total RNA isolated from Rab8a KO and wild-type (litter-mate) mice jejunal mucosa using mouse Rab8a and β-actin gene-specific primers. Data are mean ± SE of at least three separate samples from three different mice. *p < 0.01. F, Top) Western blot analysis was performed on Rab8a KO (right) and wild-type litter-mate (left) mouse jejunum mucosal (60 μg) proteins. Blots were incubated with rabbit polyclonal anti-mouse Rab8a antibodies along with β-actin antibodies. Bottom, densitometric values. Data are mean ± SE of at least three sets of samples from three different mice.

Article Snippet: For uptake, mRNA, and western blot analysis, Caco-2 cells were grown on 12 well plats (Corning Inc., NY) and Rab8a siRNA [pool of three different siRNA duplexes (duplex-1, sense: GAACUGGAUUCGCAACAUUtt, antisense: AAUGUUGCGAAUCCAGUUCtt; duplex-2, sense: GAACAAGUGUGAUGUGAAUtt, antisense: AUUCACAUCACACUUGUUCtt; duplex-3, sense: CCAGAAUGCAAUUGAGAAAtt, antisense: UUUCUCAAUUGCAUUCUGGtt; specific for human Rab8a (Santa Cruz, CA)] was transfected at 80% confluence.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Isolation, Control, Western Blot, Incubation

Journal: Digestive diseases and sciences

Article Title: Modulation of function of sodium-dependent vitamin C transporter 1 (SVCT1) by Rab8a in intestinal epithelial cells: Studies utilizing Caco-2 cells and Rab8a knockout mice

doi: 10.1007/s10620-012-2388-9

Figure Lengend Snippet: A) Real-time PCR was performed using primers for hSVCT1, hSVCT2 and β-actin on total RNA isolated from control (scrambled) and Rab8a siRNAs treated Caco-2 cells. Data are mean ± SE of multiple experiments performed on different batches of cells. B, Top, western blot analysis was performed on cell extract (60 μg) isolated from control and Rab8a siRNAs treated Caco-2 cells. Blots were incubated with rabbit anti-human hSVCT1 specific antibodies along with β-actin antibodies. Bottom, densitometric values. Data are mean ± SE of multiple experiments performed on separately isolated samples.*p < 0.01. C) Real-time PCR was performed on total RNA isolated from Rab8a KO and wild-type litter-mate mice jejunal mucosa using mouse SVCT1, SVCT2 and β-actin primers. Data are mean ± SE of at least three separate samples from three different mice. D, Top, western blot analysis was performed on Rab8a KO and wild-type (litter-mate) mouse jejunum mucosal (60 μg) proteins. Blots were incubated with rabbit anti-mouse SVCT1 antibodies along with β-actin antibodies. Bottom, densitometric values. Data are mean ± SE of at least three separate samples from three mice. *p < 0.02.

Article Snippet: For uptake, mRNA, and western blot analysis, Caco-2 cells were grown on 12 well plats (Corning Inc., NY) and Rab8a siRNA [pool of three different siRNA duplexes (duplex-1, sense: GAACUGGAUUCGCAACAUUtt, antisense: AAUGUUGCGAAUCCAGUUCtt; duplex-2, sense: GAACAAGUGUGAUGUGAAUtt, antisense: AUUCACAUCACACUUGUUCtt; duplex-3, sense: CCAGAAUGCAAUUGAGAAAtt, antisense: UUUCUCAAUUGCAUUCUGGtt; specific for human Rab8a (Santa Cruz, CA)] was transfected at 80% confluence.

Techniques: Real-time Polymerase Chain Reaction, Isolation, Control, Western Blot, Incubation

Journal: Digestive diseases and sciences

Article Title: Modulation of function of sodium-dependent vitamin C transporter 1 (SVCT1) by Rab8a in intestinal epithelial cells: Studies utilizing Caco-2 cells and Rab8a knockout mice

doi: 10.1007/s10620-012-2388-9

Figure Lengend Snippet: Caco-2 cells were transiently transfected with Rab8a siRNA or control siRNA. Forty eight hours after transfection cells were processed for biotinylation. Equal amount of protein was loaded onto pre-made 4-12% mini-gel and western blot was performed using anti-hSVCT1 antibody. The level of cell surface expression was normalized relative to the total amount of cellular hSVCT1 protein. Densitometric values are from mean ± SE of four independent experiments. Inset shows representative western blot images. *p < 0.01.

Article Snippet: For uptake, mRNA, and western blot analysis, Caco-2 cells were grown on 12 well plats (Corning Inc., NY) and Rab8a siRNA [pool of three different siRNA duplexes (duplex-1, sense: GAACUGGAUUCGCAACAUUtt, antisense: AAUGUUGCGAAUCCAGUUCtt; duplex-2, sense: GAACAAGUGUGAUGUGAAUtt, antisense: AUUCACAUCACACUUGUUCtt; duplex-3, sense: CCAGAAUGCAAUUGAGAAAtt, antisense: UUUCUCAAUUGCAUUCUGGtt; specific for human Rab8a (Santa Cruz, CA)] was transfected at 80% confluence.

Techniques: Transfection, Control, Western Blot, Expressing

Journal: Digestive diseases and sciences

Article Title: Modulation of function of sodium-dependent vitamin C transporter 1 (SVCT1) by Rab8a in intestinal epithelial cells: Studies utilizing Caco-2 cells and Rab8a knockout mice

doi: 10.1007/s10620-012-2388-9

Figure Lengend Snippet: Hu-Tu-80 cells were co-transfected with hSVCT1-YEP, LAMP1-RFP, control or Rab8a siRNAs. Live cell confocal imaging (laterl sections-xy) was performed after 48 h of transfection. Data are from n > 6-10 transfected cells and representative images were shown. Scale bar is 10 μm.

Article Snippet: For uptake, mRNA, and western blot analysis, Caco-2 cells were grown on 12 well plats (Corning Inc., NY) and Rab8a siRNA [pool of three different siRNA duplexes (duplex-1, sense: GAACUGGAUUCGCAACAUUtt, antisense: AAUGUUGCGAAUCCAGUUCtt; duplex-2, sense: GAACAAGUGUGAUGUGAAUtt, antisense: AUUCACAUCACACUUGUUCtt; duplex-3, sense: CCAGAAUGCAAUUGAGAAAtt, antisense: UUUCUCAAUUGCAUUCUGGtt; specific for human Rab8a (Santa Cruz, CA)] was transfected at 80% confluence.

Techniques: Transfection, Control, Imaging