rabbit polyclonal antibodies  (Alomone Labs)


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    Structured Review

    Alomone Labs rabbit polyclonal antibodies
    Unglycosylated β2 is retained in the ER. MDCK cells were transiently transfected with the SCN2B-yfp vector to express WT or fully unglycosylated β2 (ung) and grown for 1 day in wells. Cells were treated with TUN 2 h after transfection or left untreated (−), fixed, and immunostained with a rabbit <t>polyclonal</t> antibody against calnexin (red). A, representative xy sections show that unglycosylated β2 (green) is intracellular and overlaps with calnexin, as does the WT in TUN-treated cells. This contrasts with the localization of β2 WT at the cell end in untreated cells, also displaying a scattered pattern. To focus more accurately where β2 is found in each condition, sections were taken right above the nucleus (WT −) or at the nuclear level (for the rest). Nuclear staining by DAPI is in blue. Scale bar, 10 μm. B, line chart showing Manders' coefficients calculated along the z axis and indicating the fraction of β2 overlapping to compartments labeled with calnexin. The high overlap in TUN-treated cells and in those expressing unglycosylated β2 contrasts with negligible overlap in untreated cells expressing β2 WT. One-way ANOVA with Tukey's HSD post hoc test revealed differences among means (*, p < 0.0005). Data are mean ± S.D. (error bars) (n ≥ 3).
    Rabbit Polyclonal Antibodies, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit polyclonal antibodies/product/Alomone Labs
    Average 91 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit polyclonal antibodies - by Bioz Stars, 2023-01
    91/100 stars

    Images

    1) Product Images from "N -Glycosylation of the voltage-gated sodium channel β2 subunit is required for efficient trafficking of Na V 1.5/β2 to the plasma membrane"

    Article Title: N -Glycosylation of the voltage-gated sodium channel β2 subunit is required for efficient trafficking of Na V 1.5/β2 to the plasma membrane

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA119.007903

    Unglycosylated β2 is retained in the ER. MDCK cells were transiently transfected with the SCN2B-yfp vector to express WT or fully unglycosylated β2 (ung) and grown for 1 day in wells. Cells were treated with TUN 2 h after transfection or left untreated (−), fixed, and immunostained with a rabbit polyclonal antibody against calnexin (red). A, representative xy sections show that unglycosylated β2 (green) is intracellular and overlaps with calnexin, as does the WT in TUN-treated cells. This contrasts with the localization of β2 WT at the cell end in untreated cells, also displaying a scattered pattern. To focus more accurately where β2 is found in each condition, sections were taken right above the nucleus (WT −) or at the nuclear level (for the rest). Nuclear staining by DAPI is in blue. Scale bar, 10 μm. B, line chart showing Manders' coefficients calculated along the z axis and indicating the fraction of β2 overlapping to compartments labeled with calnexin. The high overlap in TUN-treated cells and in those expressing unglycosylated β2 contrasts with negligible overlap in untreated cells expressing β2 WT. One-way ANOVA with Tukey's HSD post hoc test revealed differences among means (*, p < 0.0005). Data are mean ± S.D. (error bars) (n ≥ 3).
    Figure Legend Snippet: Unglycosylated β2 is retained in the ER. MDCK cells were transiently transfected with the SCN2B-yfp vector to express WT or fully unglycosylated β2 (ung) and grown for 1 day in wells. Cells were treated with TUN 2 h after transfection or left untreated (−), fixed, and immunostained with a rabbit polyclonal antibody against calnexin (red). A, representative xy sections show that unglycosylated β2 (green) is intracellular and overlaps with calnexin, as does the WT in TUN-treated cells. This contrasts with the localization of β2 WT at the cell end in untreated cells, also displaying a scattered pattern. To focus more accurately where β2 is found in each condition, sections were taken right above the nucleus (WT −) or at the nuclear level (for the rest). Nuclear staining by DAPI is in blue. Scale bar, 10 μm. B, line chart showing Manders' coefficients calculated along the z axis and indicating the fraction of β2 overlapping to compartments labeled with calnexin. The high overlap in TUN-treated cells and in those expressing unglycosylated β2 contrasts with negligible overlap in untreated cells expressing β2 WT. One-way ANOVA with Tukey's HSD post hoc test revealed differences among means (*, p < 0.0005). Data are mean ± S.D. (error bars) (n ≥ 3).

    Techniques Used: Transfection, Plasmid Preparation, Staining, Labeling, Expressing

    BFA prevents complex glycosylation of β2, a fraction of which can reach the cell surface. MDCK cells were transiently transfected with the SCN2B-yfp vector to express WT or fully unglycosylated β2 (ung) and then treated 2 h later with BFA (+) or left untreated (−) and grown overnight in wells. A and C, cells were surface-biotinylated at 4 °C. The same amount of protein was used to process each lysate (∼100 μg), and the corresponding portion (nine-tenths) was subjected to overnight pulldown. Denatured protein from cell lysates and pulldowns was treated overnight at 37 °C with Endo H to cleave off immature N-glycans or left untreated (−). Representative Western blots show that the (lower) faster-migrating band of β2 WT is the only one visible in cells treated with BFA and increases its mobility with the Endo H treatment; this band coincides with unglycosylated β2 (C; compare with Fig. 2A). Note that Endo H digestion in pulldowns is only partial, either due to saturation of the enzyme or to suboptimal conditions for enzyme action. Blots for Na/K-ATPase are included as loading controls. Molecular mass markers are in kDa. B, band quantitation shows reduced levels of immature β2 in biotin-NeutrAvidin pulldowns (Membrane) of BFA-treated cells. Two-tailed Student's t test shows significant difference (*, p < 0.05). Data are mean ± S.D. (error bars) (n ≥ 3). D, cells were fixed and immunostained with a rabbit polyclonal antibody against calnexin (red) and a mouse monoclonal to GM130 (blue). Representative xy sections show that, in BFA-treated cells, β2 WT displays an intracellular accumulation comparable with mutated β2 (green), grossly overlapping with calnexin in enlarged structures (arrowheads). This contrasts with its apparent localization in the plasma membrane in untreated cells, displaying also a scattered pattern that does not overlap with calnexin (sections were taken at the cell level where β2 is mainly found in each case). Nuclear staining by DAPI is shown in gray. Scale bar, 10 μm.
    Figure Legend Snippet: BFA prevents complex glycosylation of β2, a fraction of which can reach the cell surface. MDCK cells were transiently transfected with the SCN2B-yfp vector to express WT or fully unglycosylated β2 (ung) and then treated 2 h later with BFA (+) or left untreated (−) and grown overnight in wells. A and C, cells were surface-biotinylated at 4 °C. The same amount of protein was used to process each lysate (∼100 μg), and the corresponding portion (nine-tenths) was subjected to overnight pulldown. Denatured protein from cell lysates and pulldowns was treated overnight at 37 °C with Endo H to cleave off immature N-glycans or left untreated (−). Representative Western blots show that the (lower) faster-migrating band of β2 WT is the only one visible in cells treated with BFA and increases its mobility with the Endo H treatment; this band coincides with unglycosylated β2 (C; compare with Fig. 2A). Note that Endo H digestion in pulldowns is only partial, either due to saturation of the enzyme or to suboptimal conditions for enzyme action. Blots for Na/K-ATPase are included as loading controls. Molecular mass markers are in kDa. B, band quantitation shows reduced levels of immature β2 in biotin-NeutrAvidin pulldowns (Membrane) of BFA-treated cells. Two-tailed Student's t test shows significant difference (*, p < 0.05). Data are mean ± S.D. (error bars) (n ≥ 3). D, cells were fixed and immunostained with a rabbit polyclonal antibody against calnexin (red) and a mouse monoclonal to GM130 (blue). Representative xy sections show that, in BFA-treated cells, β2 WT displays an intracellular accumulation comparable with mutated β2 (green), grossly overlapping with calnexin in enlarged structures (arrowheads). This contrasts with its apparent localization in the plasma membrane in untreated cells, displaying also a scattered pattern that does not overlap with calnexin (sections were taken at the cell level where β2 is mainly found in each case). Nuclear staining by DAPI is shown in gray. Scale bar, 10 μm.

    Techniques Used: Transfection, Plasmid Preparation, Western Blot, Quantitation Assay, Two Tailed Test, Staining

    Surface localization of NaV1.5 is reduced with unglycosylated β2. A and B, MDCK cells stably expressing WT or fully unglycosylated (ung) β2-YFP were transiently transfected with the vector SCN5A-FLAG and grown polarized in Transwells. Cells were fixed and immunostained with a rabbit polyclonal antibody against NaV1.5 (red) and with a mouse mAb to gp114 (cyan). Images were obtained by confocal microscopy. In merged images, the YFP-emitted fluorescence is shown in green and DAPI is in blue. Representative xy sections taken at the apical (A) or nuclear (B) levels (sections taken at the cell level where NaV1.5 is mainly found in each case) and corresponding z axis reconstruction (reciprocal xz and xy sections marked by a yellow dashed line) show improved apical localization of NaV1.5 with β2 WT (A), which remains mostly intracellular in the presence of unglycosylated β2 (B); note the intracellular NaV1.5 accumulation with mutated β2 (arrowhead). Scale bars, 10 μm. C and D, line charts displaying the CTCF (mean percentage ± S.D. (error bars)) along an apical-to-basal z-stack (section 1: most apical; 0.5-μm optical slice thickness) show the NaV1.5 curve peak close to those of apical gp114 and β2 WT (C). In contrast, NaV1.5 is displaced toward the nuclear section with mutated β2, which overlays with DAPI (D), included as reference for the nuclear level (≥6 cells were analyzed per condition). E, MDCK cells stably expressing NaV1.5-YFP were transiently cotransfected with the SCN2B-yfp vector to express β2, WT or fully unglycosylated (ung), plus additional SCN5A-FLAG vector to ensure extensive NaV1.5 overexpression, and grown overnight in wells; the pEGFP-N1 vector was used as a control. Cells were surface-biotinylated at 4 °C. The same amount of protein was used to process each lysate (∼600 μg), 97% of which was subjected to overnight NeutrAvidin pulldown. Representative Western blots and band quantitation (F) show reduced levels of NaV1.5 in biotin-NeutrAvidin pulldowns (Membrane) in the presence of unglycosylated β2 or without β2 (GFP), when comparing with the WT. One-way ANOVA with Tukey's HSD post hoc test showed significant differences (*, p < 0.002). The percentage of NaV1.5 at the cell surface over total cellular NaV1.5 protein varied from 1.42 ± 0.98 in the WT to 0.73 ± 0.50% with unglycosylated β2. Data are mean ± S.D. (n ≥ 6). Na/K-ATPase was blotted as surface marker to correct for quantitations in pulldowns. Molecular mass markers are in kDa. For clear display, the blot in E shows lysates and pulldowns separated by division lines, which indicate different exposure between lysates and pulldowns but equal exposure within each group.
    Figure Legend Snippet: Surface localization of NaV1.5 is reduced with unglycosylated β2. A and B, MDCK cells stably expressing WT or fully unglycosylated (ung) β2-YFP were transiently transfected with the vector SCN5A-FLAG and grown polarized in Transwells. Cells were fixed and immunostained with a rabbit polyclonal antibody against NaV1.5 (red) and with a mouse mAb to gp114 (cyan). Images were obtained by confocal microscopy. In merged images, the YFP-emitted fluorescence is shown in green and DAPI is in blue. Representative xy sections taken at the apical (A) or nuclear (B) levels (sections taken at the cell level where NaV1.5 is mainly found in each case) and corresponding z axis reconstruction (reciprocal xz and xy sections marked by a yellow dashed line) show improved apical localization of NaV1.5 with β2 WT (A), which remains mostly intracellular in the presence of unglycosylated β2 (B); note the intracellular NaV1.5 accumulation with mutated β2 (arrowhead). Scale bars, 10 μm. C and D, line charts displaying the CTCF (mean percentage ± S.D. (error bars)) along an apical-to-basal z-stack (section 1: most apical; 0.5-μm optical slice thickness) show the NaV1.5 curve peak close to those of apical gp114 and β2 WT (C). In contrast, NaV1.5 is displaced toward the nuclear section with mutated β2, which overlays with DAPI (D), included as reference for the nuclear level (≥6 cells were analyzed per condition). E, MDCK cells stably expressing NaV1.5-YFP were transiently cotransfected with the SCN2B-yfp vector to express β2, WT or fully unglycosylated (ung), plus additional SCN5A-FLAG vector to ensure extensive NaV1.5 overexpression, and grown overnight in wells; the pEGFP-N1 vector was used as a control. Cells were surface-biotinylated at 4 °C. The same amount of protein was used to process each lysate (∼600 μg), 97% of which was subjected to overnight NeutrAvidin pulldown. Representative Western blots and band quantitation (F) show reduced levels of NaV1.5 in biotin-NeutrAvidin pulldowns (Membrane) in the presence of unglycosylated β2 or without β2 (GFP), when comparing with the WT. One-way ANOVA with Tukey's HSD post hoc test showed significant differences (*, p < 0.002). The percentage of NaV1.5 at the cell surface over total cellular NaV1.5 protein varied from 1.42 ± 0.98 in the WT to 0.73 ± 0.50% with unglycosylated β2. Data are mean ± S.D. (n ≥ 6). Na/K-ATPase was blotted as surface marker to correct for quantitations in pulldowns. Molecular mass markers are in kDa. For clear display, the blot in E shows lysates and pulldowns separated by division lines, which indicate different exposure between lysates and pulldowns but equal exposure within each group.

    Techniques Used: Stable Transfection, Expressing, Transfection, Plasmid Preparation, Confocal Microscopy, Fluorescence, Over Expression, Western Blot, Quantitation Assay, Marker

    Defect over time of unglycosylated β2 in promoting surface localization of NaV1.5. MDCK cells stably expressing WT (A) or fully unglycosylated (ung) β2-YFP (C) or untransfected (parental) cells (E) were transiently transfected with the vector SCN5A-FLAG and grown in wells for the indicated number of days. Cells were fixed and immunostained with a rabbit polyclonal antibody against NaV1.5 (red) and with a mouse mAb to Na/K-ATPase (blue). Images were obtained by confocal microscopy. In merged images, the YFP-emitted fluorescence is shown in green and DAPI is in gray. A, C, and E, representative xy sections (sections taken at the cell level where NaV1.5 is mainly found in each case) show a general diffuse NaV1.5 pattern, intracellular and often perinuclear, except for a noticeable overlap with Na/K-ATPase, particularly at day 1, in the presence of β2 WT. Scale bars, 10 μm. Confocal images were analyzed by calculating the MFI along linear segments of 30 pixels in length (d, distance; 0.1 μm/pixel) drawn from the cell end perpendicularly into the cytoplasm. B, D, and F, line charts show MFIs with the first 5 pixels of the segments, equivalent to the plasma membrane region (cell end), marked with a square bracket. The highest MFI levels are at the cell end for Na/K-ATPase and for β2 WT, which progressively decrease intracellularly. The profile for NaV1.5 increases at the cell end only in the presence of β2 WT and especially at day 1 (B) but remains comparatively low within this region with unglycosylated β2 (D) or in the absence of β2 (F). Data are mean ± S.D. (error bars) (number of cells analyzed ≥3; 4 segments/cell).
    Figure Legend Snippet: Defect over time of unglycosylated β2 in promoting surface localization of NaV1.5. MDCK cells stably expressing WT (A) or fully unglycosylated (ung) β2-YFP (C) or untransfected (parental) cells (E) were transiently transfected with the vector SCN5A-FLAG and grown in wells for the indicated number of days. Cells were fixed and immunostained with a rabbit polyclonal antibody against NaV1.5 (red) and with a mouse mAb to Na/K-ATPase (blue). Images were obtained by confocal microscopy. In merged images, the YFP-emitted fluorescence is shown in green and DAPI is in gray. A, C, and E, representative xy sections (sections taken at the cell level where NaV1.5 is mainly found in each case) show a general diffuse NaV1.5 pattern, intracellular and often perinuclear, except for a noticeable overlap with Na/K-ATPase, particularly at day 1, in the presence of β2 WT. Scale bars, 10 μm. Confocal images were analyzed by calculating the MFI along linear segments of 30 pixels in length (d, distance; 0.1 μm/pixel) drawn from the cell end perpendicularly into the cytoplasm. B, D, and F, line charts show MFIs with the first 5 pixels of the segments, equivalent to the plasma membrane region (cell end), marked with a square bracket. The highest MFI levels are at the cell end for Na/K-ATPase and for β2 WT, which progressively decrease intracellularly. The profile for NaV1.5 increases at the cell end only in the presence of β2 WT and especially at day 1 (B) but remains comparatively low within this region with unglycosylated β2 (D) or in the absence of β2 (F). Data are mean ± S.D. (error bars) (number of cells analyzed ≥3; 4 segments/cell).

    Techniques Used: Stable Transfection, Expressing, Transfection, Plasmid Preparation, Confocal Microscopy, Fluorescence

    Single β2 glycosylation mutants can promote surface localization of NaV1.5; analysis over time. MDCK cells stably expressing the indicated single mutant for β2-YFP glycosylation were transiently transfected with the vector SCN5A-FLAG and grown in wells for the specified number of days. Cells were fixed and immunostained with a rabbit polyclonal antibody against NaV1.5 (red) and with a mouse mAb to Na/K-ATPase (blue). Images were obtained by confocal microscopy. In merged images, the YFP-emitted fluorescence is in green and DAPI is in gray. A, C, and E, Representative xy sections (taken at the level where NaV1.5 is mainly found in each case) show some areas of overlap of NaV1.5 with Na/K-ATPase at the cell end, particularly at day 1, in the presence of any of the mutants, while remaining mostly disperse throughout the cell at later time points. Scale bars, 10 μm. Confocal images were analyzed by calculating the MFI along linear segments of 30 pixels in length (d, distance; 0.1 μm/pixel) drawn from the cell end perpendicularly into the cytoplasm. B, D, and F, line charts show MFIs with the first 5 pixels of the segments, equivalent to the plasma membrane region (cell end), marked with a square bracket. The highest MFI levels are at the cell end for Na/K-ATPase and for the different β2 single mutants, all progressively decreasing intracellularly. The profile for NaV1.5 increases at the cell end at day 1 in all cases, remaining comparatively low within this region at later time points. Data are mean ± S.D. (error bars) (number of cells analyzed ≥3, 4 segments/cell).
    Figure Legend Snippet: Single β2 glycosylation mutants can promote surface localization of NaV1.5; analysis over time. MDCK cells stably expressing the indicated single mutant for β2-YFP glycosylation were transiently transfected with the vector SCN5A-FLAG and grown in wells for the specified number of days. Cells were fixed and immunostained with a rabbit polyclonal antibody against NaV1.5 (red) and with a mouse mAb to Na/K-ATPase (blue). Images were obtained by confocal microscopy. In merged images, the YFP-emitted fluorescence is in green and DAPI is in gray. A, C, and E, Representative xy sections (taken at the level where NaV1.5 is mainly found in each case) show some areas of overlap of NaV1.5 with Na/K-ATPase at the cell end, particularly at day 1, in the presence of any of the mutants, while remaining mostly disperse throughout the cell at later time points. Scale bars, 10 μm. Confocal images were analyzed by calculating the MFI along linear segments of 30 pixels in length (d, distance; 0.1 μm/pixel) drawn from the cell end perpendicularly into the cytoplasm. B, D, and F, line charts show MFIs with the first 5 pixels of the segments, equivalent to the plasma membrane region (cell end), marked with a square bracket. The highest MFI levels are at the cell end for Na/K-ATPase and for the different β2 single mutants, all progressively decreasing intracellularly. The profile for NaV1.5 increases at the cell end at day 1 in all cases, remaining comparatively low within this region at later time points. Data are mean ± S.D. (error bars) (number of cells analyzed ≥3, 4 segments/cell).

    Techniques Used: Stable Transfection, Expressing, Mutagenesis, Transfection, Plasmid Preparation, Confocal Microscopy, Fluorescence

    Single and double glycosylation mutants of β2 can promote surface localization of NaV1.5. MDCK cells (A, C, and E) stably expressing the indicated single mutant for β2-YFP glycosylation were transiently transfected with the vector SCN5A-FLAG and grown polarized in Transwells. Cells were fixed and immunostained with a rabbit polyclonal antibody against NaV1.5 (red), and with a mouse mAb to gp114 (cyan). Images were obtained by confocal microscopy. In merged images, the YFP-emitted fluorescence is in green, and DAPI is in gray. Representative xy sections taken at the apical level (section level chosen to assess presence of NaV1.5 at the apical surface) and corresponding z axis reconstruction (reciprocal xz and xy sections marked by a yellow dashed line) show noticeable apical localization of NaV1.5 with the different β2 variants. Scale bars, 10 μm. B, D, and F, line charts displaying the CTCF (mean percentage ± S.D. (error bars)) along an apical-to-basal z-stack (section 1: most apical; 0.5-μm optical slice thickness) show the NaV1.5 curve peak in close proximity to those of apical gp114 and any of the β2 mutants. DAPI is included as reference for the nuclear level (≥6 cells were analyzed per condition). G and I, MDCK cells stably expressing NaV1.5-YFP were transiently cotransfected with the SCN2B-yfp vector to express β2-YFP, WT, or any of the indicated single (G) or double (I) mutants, plus additional SCN5A-FLAG vector to ensure extensive NaV1.5 overexpression, and grown overnight in wells. Cells were surface-biotinylated at 4 °C. The same amount of protein was used to process each lysate (∼600 μg), 97% of which was subjected to overnight NeutrAvidin pulldown. Representative Western blots (G and I) and band quantitation (H and J) show comparable levels of NaV1.5 in biotin-NeutrAvidin pulldowns (Membrane) in the presence of any mutant variant of β2 as with the WT. One-way ANOVA revealed no differences among means. Data are mean ± S.D. (n ≥ 3). Na/K-ATPase was blotted as surface marker to correct for quantitations in pulldowns. Molecular mass markers are in kDa. For clear display, the blots in G and I show lysates and pulldowns separated by division lines, which indicate different exposure between each.
    Figure Legend Snippet: Single and double glycosylation mutants of β2 can promote surface localization of NaV1.5. MDCK cells (A, C, and E) stably expressing the indicated single mutant for β2-YFP glycosylation were transiently transfected with the vector SCN5A-FLAG and grown polarized in Transwells. Cells were fixed and immunostained with a rabbit polyclonal antibody against NaV1.5 (red), and with a mouse mAb to gp114 (cyan). Images were obtained by confocal microscopy. In merged images, the YFP-emitted fluorescence is in green, and DAPI is in gray. Representative xy sections taken at the apical level (section level chosen to assess presence of NaV1.5 at the apical surface) and corresponding z axis reconstruction (reciprocal xz and xy sections marked by a yellow dashed line) show noticeable apical localization of NaV1.5 with the different β2 variants. Scale bars, 10 μm. B, D, and F, line charts displaying the CTCF (mean percentage ± S.D. (error bars)) along an apical-to-basal z-stack (section 1: most apical; 0.5-μm optical slice thickness) show the NaV1.5 curve peak in close proximity to those of apical gp114 and any of the β2 mutants. DAPI is included as reference for the nuclear level (≥6 cells were analyzed per condition). G and I, MDCK cells stably expressing NaV1.5-YFP were transiently cotransfected with the SCN2B-yfp vector to express β2-YFP, WT, or any of the indicated single (G) or double (I) mutants, plus additional SCN5A-FLAG vector to ensure extensive NaV1.5 overexpression, and grown overnight in wells. Cells were surface-biotinylated at 4 °C. The same amount of protein was used to process each lysate (∼600 μg), 97% of which was subjected to overnight NeutrAvidin pulldown. Representative Western blots (G and I) and band quantitation (H and J) show comparable levels of NaV1.5 in biotin-NeutrAvidin pulldowns (Membrane) in the presence of any mutant variant of β2 as with the WT. One-way ANOVA revealed no differences among means. Data are mean ± S.D. (n ≥ 3). Na/K-ATPase was blotted as surface marker to correct for quantitations in pulldowns. Molecular mass markers are in kDa. For clear display, the blots in G and I show lysates and pulldowns separated by division lines, which indicate different exposure between each.

    Techniques Used: Stable Transfection, Expressing, Mutagenesis, Transfection, Plasmid Preparation, Confocal Microscopy, Fluorescence, Over Expression, Western Blot, Quantitation Assay, Variant Assay, Marker

    rabbit polyclonal antibodies  (Alomone Labs)


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    Structured Review

    Alomone Labs rabbit polyclonal antibodies
    Unglycosylated β2 is retained in the ER. MDCK cells were transiently transfected with the SCN2B-yfp vector to express WT or fully unglycosylated β2 (ung) and grown for 1 day in wells. Cells were treated with TUN 2 h after transfection or left untreated (−), fixed, and immunostained with a rabbit <t>polyclonal</t> antibody against calnexin (red). A, representative xy sections show that unglycosylated β2 (green) is intracellular and overlaps with calnexin, as does the WT in TUN-treated cells. This contrasts with the localization of β2 WT at the cell end in untreated cells, also displaying a scattered pattern. To focus more accurately where β2 is found in each condition, sections were taken right above the nucleus (WT −) or at the nuclear level (for the rest). Nuclear staining by DAPI is in blue. Scale bar, 10 μm. B, line chart showing Manders' coefficients calculated along the z axis and indicating the fraction of β2 overlapping to compartments labeled with calnexin. The high overlap in TUN-treated cells and in those expressing unglycosylated β2 contrasts with negligible overlap in untreated cells expressing β2 WT. One-way ANOVA with Tukey's HSD post hoc test revealed differences among means (*, p < 0.0005). Data are mean ± S.D. (error bars) (n ≥ 3).
    Rabbit Polyclonal Antibodies, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit polyclonal antibodies/product/Alomone Labs
    Average 91 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit polyclonal antibodies - by Bioz Stars, 2023-01
    91/100 stars

    Images

    1) Product Images from "N -Glycosylation of the voltage-gated sodium channel β2 subunit is required for efficient trafficking of Na V 1.5/β2 to the plasma membrane"

    Article Title: N -Glycosylation of the voltage-gated sodium channel β2 subunit is required for efficient trafficking of Na V 1.5/β2 to the plasma membrane

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA119.007903

    Unglycosylated β2 is retained in the ER. MDCK cells were transiently transfected with the SCN2B-yfp vector to express WT or fully unglycosylated β2 (ung) and grown for 1 day in wells. Cells were treated with TUN 2 h after transfection or left untreated (−), fixed, and immunostained with a rabbit polyclonal antibody against calnexin (red). A, representative xy sections show that unglycosylated β2 (green) is intracellular and overlaps with calnexin, as does the WT in TUN-treated cells. This contrasts with the localization of β2 WT at the cell end in untreated cells, also displaying a scattered pattern. To focus more accurately where β2 is found in each condition, sections were taken right above the nucleus (WT −) or at the nuclear level (for the rest). Nuclear staining by DAPI is in blue. Scale bar, 10 μm. B, line chart showing Manders' coefficients calculated along the z axis and indicating the fraction of β2 overlapping to compartments labeled with calnexin. The high overlap in TUN-treated cells and in those expressing unglycosylated β2 contrasts with negligible overlap in untreated cells expressing β2 WT. One-way ANOVA with Tukey's HSD post hoc test revealed differences among means (*, p < 0.0005). Data are mean ± S.D. (error bars) (n ≥ 3).
    Figure Legend Snippet: Unglycosylated β2 is retained in the ER. MDCK cells were transiently transfected with the SCN2B-yfp vector to express WT or fully unglycosylated β2 (ung) and grown for 1 day in wells. Cells were treated with TUN 2 h after transfection or left untreated (−), fixed, and immunostained with a rabbit polyclonal antibody against calnexin (red). A, representative xy sections show that unglycosylated β2 (green) is intracellular and overlaps with calnexin, as does the WT in TUN-treated cells. This contrasts with the localization of β2 WT at the cell end in untreated cells, also displaying a scattered pattern. To focus more accurately where β2 is found in each condition, sections were taken right above the nucleus (WT −) or at the nuclear level (for the rest). Nuclear staining by DAPI is in blue. Scale bar, 10 μm. B, line chart showing Manders' coefficients calculated along the z axis and indicating the fraction of β2 overlapping to compartments labeled with calnexin. The high overlap in TUN-treated cells and in those expressing unglycosylated β2 contrasts with negligible overlap in untreated cells expressing β2 WT. One-way ANOVA with Tukey's HSD post hoc test revealed differences among means (*, p < 0.0005). Data are mean ± S.D. (error bars) (n ≥ 3).

    Techniques Used: Transfection, Plasmid Preparation, Staining, Labeling, Expressing

    BFA prevents complex glycosylation of β2, a fraction of which can reach the cell surface. MDCK cells were transiently transfected with the SCN2B-yfp vector to express WT or fully unglycosylated β2 (ung) and then treated 2 h later with BFA (+) or left untreated (−) and grown overnight in wells. A and C, cells were surface-biotinylated at 4 °C. The same amount of protein was used to process each lysate (∼100 μg), and the corresponding portion (nine-tenths) was subjected to overnight pulldown. Denatured protein from cell lysates and pulldowns was treated overnight at 37 °C with Endo H to cleave off immature N-glycans or left untreated (−). Representative Western blots show that the (lower) faster-migrating band of β2 WT is the only one visible in cells treated with BFA and increases its mobility with the Endo H treatment; this band coincides with unglycosylated β2 (C; compare with Fig. 2A). Note that Endo H digestion in pulldowns is only partial, either due to saturation of the enzyme or to suboptimal conditions for enzyme action. Blots for Na/K-ATPase are included as loading controls. Molecular mass markers are in kDa. B, band quantitation shows reduced levels of immature β2 in biotin-NeutrAvidin pulldowns (Membrane) of BFA-treated cells. Two-tailed Student's t test shows significant difference (*, p < 0.05). Data are mean ± S.D. (error bars) (n ≥ 3). D, cells were fixed and immunostained with a rabbit polyclonal antibody against calnexin (red) and a mouse monoclonal to GM130 (blue). Representative xy sections show that, in BFA-treated cells, β2 WT displays an intracellular accumulation comparable with mutated β2 (green), grossly overlapping with calnexin in enlarged structures (arrowheads). This contrasts with its apparent localization in the plasma membrane in untreated cells, displaying also a scattered pattern that does not overlap with calnexin (sections were taken at the cell level where β2 is mainly found in each case). Nuclear staining by DAPI is shown in gray. Scale bar, 10 μm.
    Figure Legend Snippet: BFA prevents complex glycosylation of β2, a fraction of which can reach the cell surface. MDCK cells were transiently transfected with the SCN2B-yfp vector to express WT or fully unglycosylated β2 (ung) and then treated 2 h later with BFA (+) or left untreated (−) and grown overnight in wells. A and C, cells were surface-biotinylated at 4 °C. The same amount of protein was used to process each lysate (∼100 μg), and the corresponding portion (nine-tenths) was subjected to overnight pulldown. Denatured protein from cell lysates and pulldowns was treated overnight at 37 °C with Endo H to cleave off immature N-glycans or left untreated (−). Representative Western blots show that the (lower) faster-migrating band of β2 WT is the only one visible in cells treated with BFA and increases its mobility with the Endo H treatment; this band coincides with unglycosylated β2 (C; compare with Fig. 2A). Note that Endo H digestion in pulldowns is only partial, either due to saturation of the enzyme or to suboptimal conditions for enzyme action. Blots for Na/K-ATPase are included as loading controls. Molecular mass markers are in kDa. B, band quantitation shows reduced levels of immature β2 in biotin-NeutrAvidin pulldowns (Membrane) of BFA-treated cells. Two-tailed Student's t test shows significant difference (*, p < 0.05). Data are mean ± S.D. (error bars) (n ≥ 3). D, cells were fixed and immunostained with a rabbit polyclonal antibody against calnexin (red) and a mouse monoclonal to GM130 (blue). Representative xy sections show that, in BFA-treated cells, β2 WT displays an intracellular accumulation comparable with mutated β2 (green), grossly overlapping with calnexin in enlarged structures (arrowheads). This contrasts with its apparent localization in the plasma membrane in untreated cells, displaying also a scattered pattern that does not overlap with calnexin (sections were taken at the cell level where β2 is mainly found in each case). Nuclear staining by DAPI is shown in gray. Scale bar, 10 μm.

    Techniques Used: Transfection, Plasmid Preparation, Western Blot, Quantitation Assay, Two Tailed Test, Staining

    Surface localization of NaV1.5 is reduced with unglycosylated β2. A and B, MDCK cells stably expressing WT or fully unglycosylated (ung) β2-YFP were transiently transfected with the vector SCN5A-FLAG and grown polarized in Transwells. Cells were fixed and immunostained with a rabbit polyclonal antibody against NaV1.5 (red) and with a mouse mAb to gp114 (cyan). Images were obtained by confocal microscopy. In merged images, the YFP-emitted fluorescence is shown in green and DAPI is in blue. Representative xy sections taken at the apical (A) or nuclear (B) levels (sections taken at the cell level where NaV1.5 is mainly found in each case) and corresponding z axis reconstruction (reciprocal xz and xy sections marked by a yellow dashed line) show improved apical localization of NaV1.5 with β2 WT (A), which remains mostly intracellular in the presence of unglycosylated β2 (B); note the intracellular NaV1.5 accumulation with mutated β2 (arrowhead). Scale bars, 10 μm. C and D, line charts displaying the CTCF (mean percentage ± S.D. (error bars)) along an apical-to-basal z-stack (section 1: most apical; 0.5-μm optical slice thickness) show the NaV1.5 curve peak close to those of apical gp114 and β2 WT (C). In contrast, NaV1.5 is displaced toward the nuclear section with mutated β2, which overlays with DAPI (D), included as reference for the nuclear level (≥6 cells were analyzed per condition). E, MDCK cells stably expressing NaV1.5-YFP were transiently cotransfected with the SCN2B-yfp vector to express β2, WT or fully unglycosylated (ung), plus additional SCN5A-FLAG vector to ensure extensive NaV1.5 overexpression, and grown overnight in wells; the pEGFP-N1 vector was used as a control. Cells were surface-biotinylated at 4 °C. The same amount of protein was used to process each lysate (∼600 μg), 97% of which was subjected to overnight NeutrAvidin pulldown. Representative Western blots and band quantitation (F) show reduced levels of NaV1.5 in biotin-NeutrAvidin pulldowns (Membrane) in the presence of unglycosylated β2 or without β2 (GFP), when comparing with the WT. One-way ANOVA with Tukey's HSD post hoc test showed significant differences (*, p < 0.002). The percentage of NaV1.5 at the cell surface over total cellular NaV1.5 protein varied from 1.42 ± 0.98 in the WT to 0.73 ± 0.50% with unglycosylated β2. Data are mean ± S.D. (n ≥ 6). Na/K-ATPase was blotted as surface marker to correct for quantitations in pulldowns. Molecular mass markers are in kDa. For clear display, the blot in E shows lysates and pulldowns separated by division lines, which indicate different exposure between lysates and pulldowns but equal exposure within each group.
    Figure Legend Snippet: Surface localization of NaV1.5 is reduced with unglycosylated β2. A and B, MDCK cells stably expressing WT or fully unglycosylated (ung) β2-YFP were transiently transfected with the vector SCN5A-FLAG and grown polarized in Transwells. Cells were fixed and immunostained with a rabbit polyclonal antibody against NaV1.5 (red) and with a mouse mAb to gp114 (cyan). Images were obtained by confocal microscopy. In merged images, the YFP-emitted fluorescence is shown in green and DAPI is in blue. Representative xy sections taken at the apical (A) or nuclear (B) levels (sections taken at the cell level where NaV1.5 is mainly found in each case) and corresponding z axis reconstruction (reciprocal xz and xy sections marked by a yellow dashed line) show improved apical localization of NaV1.5 with β2 WT (A), which remains mostly intracellular in the presence of unglycosylated β2 (B); note the intracellular NaV1.5 accumulation with mutated β2 (arrowhead). Scale bars, 10 μm. C and D, line charts displaying the CTCF (mean percentage ± S.D. (error bars)) along an apical-to-basal z-stack (section 1: most apical; 0.5-μm optical slice thickness) show the NaV1.5 curve peak close to those of apical gp114 and β2 WT (C). In contrast, NaV1.5 is displaced toward the nuclear section with mutated β2, which overlays with DAPI (D), included as reference for the nuclear level (≥6 cells were analyzed per condition). E, MDCK cells stably expressing NaV1.5-YFP were transiently cotransfected with the SCN2B-yfp vector to express β2, WT or fully unglycosylated (ung), plus additional SCN5A-FLAG vector to ensure extensive NaV1.5 overexpression, and grown overnight in wells; the pEGFP-N1 vector was used as a control. Cells were surface-biotinylated at 4 °C. The same amount of protein was used to process each lysate (∼600 μg), 97% of which was subjected to overnight NeutrAvidin pulldown. Representative Western blots and band quantitation (F) show reduced levels of NaV1.5 in biotin-NeutrAvidin pulldowns (Membrane) in the presence of unglycosylated β2 or without β2 (GFP), when comparing with the WT. One-way ANOVA with Tukey's HSD post hoc test showed significant differences (*, p < 0.002). The percentage of NaV1.5 at the cell surface over total cellular NaV1.5 protein varied from 1.42 ± 0.98 in the WT to 0.73 ± 0.50% with unglycosylated β2. Data are mean ± S.D. (n ≥ 6). Na/K-ATPase was blotted as surface marker to correct for quantitations in pulldowns. Molecular mass markers are in kDa. For clear display, the blot in E shows lysates and pulldowns separated by division lines, which indicate different exposure between lysates and pulldowns but equal exposure within each group.

    Techniques Used: Stable Transfection, Expressing, Transfection, Plasmid Preparation, Confocal Microscopy, Fluorescence, Over Expression, Western Blot, Quantitation Assay, Marker

    Defect over time of unglycosylated β2 in promoting surface localization of NaV1.5. MDCK cells stably expressing WT (A) or fully unglycosylated (ung) β2-YFP (C) or untransfected (parental) cells (E) were transiently transfected with the vector SCN5A-FLAG and grown in wells for the indicated number of days. Cells were fixed and immunostained with a rabbit polyclonal antibody against NaV1.5 (red) and with a mouse mAb to Na/K-ATPase (blue). Images were obtained by confocal microscopy. In merged images, the YFP-emitted fluorescence is shown in green and DAPI is in gray. A, C, and E, representative xy sections (sections taken at the cell level where NaV1.5 is mainly found in each case) show a general diffuse NaV1.5 pattern, intracellular and often perinuclear, except for a noticeable overlap with Na/K-ATPase, particularly at day 1, in the presence of β2 WT. Scale bars, 10 μm. Confocal images were analyzed by calculating the MFI along linear segments of 30 pixels in length (d, distance; 0.1 μm/pixel) drawn from the cell end perpendicularly into the cytoplasm. B, D, and F, line charts show MFIs with the first 5 pixels of the segments, equivalent to the plasma membrane region (cell end), marked with a square bracket. The highest MFI levels are at the cell end for Na/K-ATPase and for β2 WT, which progressively decrease intracellularly. The profile for NaV1.5 increases at the cell end only in the presence of β2 WT and especially at day 1 (B) but remains comparatively low within this region with unglycosylated β2 (D) or in the absence of β2 (F). Data are mean ± S.D. (error bars) (number of cells analyzed ≥3; 4 segments/cell).
    Figure Legend Snippet: Defect over time of unglycosylated β2 in promoting surface localization of NaV1.5. MDCK cells stably expressing WT (A) or fully unglycosylated (ung) β2-YFP (C) or untransfected (parental) cells (E) were transiently transfected with the vector SCN5A-FLAG and grown in wells for the indicated number of days. Cells were fixed and immunostained with a rabbit polyclonal antibody against NaV1.5 (red) and with a mouse mAb to Na/K-ATPase (blue). Images were obtained by confocal microscopy. In merged images, the YFP-emitted fluorescence is shown in green and DAPI is in gray. A, C, and E, representative xy sections (sections taken at the cell level where NaV1.5 is mainly found in each case) show a general diffuse NaV1.5 pattern, intracellular and often perinuclear, except for a noticeable overlap with Na/K-ATPase, particularly at day 1, in the presence of β2 WT. Scale bars, 10 μm. Confocal images were analyzed by calculating the MFI along linear segments of 30 pixels in length (d, distance; 0.1 μm/pixel) drawn from the cell end perpendicularly into the cytoplasm. B, D, and F, line charts show MFIs with the first 5 pixels of the segments, equivalent to the plasma membrane region (cell end), marked with a square bracket. The highest MFI levels are at the cell end for Na/K-ATPase and for β2 WT, which progressively decrease intracellularly. The profile for NaV1.5 increases at the cell end only in the presence of β2 WT and especially at day 1 (B) but remains comparatively low within this region with unglycosylated β2 (D) or in the absence of β2 (F). Data are mean ± S.D. (error bars) (number of cells analyzed ≥3; 4 segments/cell).

    Techniques Used: Stable Transfection, Expressing, Transfection, Plasmid Preparation, Confocal Microscopy, Fluorescence

    Single β2 glycosylation mutants can promote surface localization of NaV1.5; analysis over time. MDCK cells stably expressing the indicated single mutant for β2-YFP glycosylation were transiently transfected with the vector SCN5A-FLAG and grown in wells for the specified number of days. Cells were fixed and immunostained with a rabbit polyclonal antibody against NaV1.5 (red) and with a mouse mAb to Na/K-ATPase (blue). Images were obtained by confocal microscopy. In merged images, the YFP-emitted fluorescence is in green and DAPI is in gray. A, C, and E, Representative xy sections (taken at the level where NaV1.5 is mainly found in each case) show some areas of overlap of NaV1.5 with Na/K-ATPase at the cell end, particularly at day 1, in the presence of any of the mutants, while remaining mostly disperse throughout the cell at later time points. Scale bars, 10 μm. Confocal images were analyzed by calculating the MFI along linear segments of 30 pixels in length (d, distance; 0.1 μm/pixel) drawn from the cell end perpendicularly into the cytoplasm. B, D, and F, line charts show MFIs with the first 5 pixels of the segments, equivalent to the plasma membrane region (cell end), marked with a square bracket. The highest MFI levels are at the cell end for Na/K-ATPase and for the different β2 single mutants, all progressively decreasing intracellularly. The profile for NaV1.5 increases at the cell end at day 1 in all cases, remaining comparatively low within this region at later time points. Data are mean ± S.D. (error bars) (number of cells analyzed ≥3, 4 segments/cell).
    Figure Legend Snippet: Single β2 glycosylation mutants can promote surface localization of NaV1.5; analysis over time. MDCK cells stably expressing the indicated single mutant for β2-YFP glycosylation were transiently transfected with the vector SCN5A-FLAG and grown in wells for the specified number of days. Cells were fixed and immunostained with a rabbit polyclonal antibody against NaV1.5 (red) and with a mouse mAb to Na/K-ATPase (blue). Images were obtained by confocal microscopy. In merged images, the YFP-emitted fluorescence is in green and DAPI is in gray. A, C, and E, Representative xy sections (taken at the level where NaV1.5 is mainly found in each case) show some areas of overlap of NaV1.5 with Na/K-ATPase at the cell end, particularly at day 1, in the presence of any of the mutants, while remaining mostly disperse throughout the cell at later time points. Scale bars, 10 μm. Confocal images were analyzed by calculating the MFI along linear segments of 30 pixels in length (d, distance; 0.1 μm/pixel) drawn from the cell end perpendicularly into the cytoplasm. B, D, and F, line charts show MFIs with the first 5 pixels of the segments, equivalent to the plasma membrane region (cell end), marked with a square bracket. The highest MFI levels are at the cell end for Na/K-ATPase and for the different β2 single mutants, all progressively decreasing intracellularly. The profile for NaV1.5 increases at the cell end at day 1 in all cases, remaining comparatively low within this region at later time points. Data are mean ± S.D. (error bars) (number of cells analyzed ≥3, 4 segments/cell).

    Techniques Used: Stable Transfection, Expressing, Mutagenesis, Transfection, Plasmid Preparation, Confocal Microscopy, Fluorescence

    Single and double glycosylation mutants of β2 can promote surface localization of NaV1.5. MDCK cells (A, C, and E) stably expressing the indicated single mutant for β2-YFP glycosylation were transiently transfected with the vector SCN5A-FLAG and grown polarized in Transwells. Cells were fixed and immunostained with a rabbit polyclonal antibody against NaV1.5 (red), and with a mouse mAb to gp114 (cyan). Images were obtained by confocal microscopy. In merged images, the YFP-emitted fluorescence is in green, and DAPI is in gray. Representative xy sections taken at the apical level (section level chosen to assess presence of NaV1.5 at the apical surface) and corresponding z axis reconstruction (reciprocal xz and xy sections marked by a yellow dashed line) show noticeable apical localization of NaV1.5 with the different β2 variants. Scale bars, 10 μm. B, D, and F, line charts displaying the CTCF (mean percentage ± S.D. (error bars)) along an apical-to-basal z-stack (section 1: most apical; 0.5-μm optical slice thickness) show the NaV1.5 curve peak in close proximity to those of apical gp114 and any of the β2 mutants. DAPI is included as reference for the nuclear level (≥6 cells were analyzed per condition). G and I, MDCK cells stably expressing NaV1.5-YFP were transiently cotransfected with the SCN2B-yfp vector to express β2-YFP, WT, or any of the indicated single (G) or double (I) mutants, plus additional SCN5A-FLAG vector to ensure extensive NaV1.5 overexpression, and grown overnight in wells. Cells were surface-biotinylated at 4 °C. The same amount of protein was used to process each lysate (∼600 μg), 97% of which was subjected to overnight NeutrAvidin pulldown. Representative Western blots (G and I) and band quantitation (H and J) show comparable levels of NaV1.5 in biotin-NeutrAvidin pulldowns (Membrane) in the presence of any mutant variant of β2 as with the WT. One-way ANOVA revealed no differences among means. Data are mean ± S.D. (n ≥ 3). Na/K-ATPase was blotted as surface marker to correct for quantitations in pulldowns. Molecular mass markers are in kDa. For clear display, the blots in G and I show lysates and pulldowns separated by division lines, which indicate different exposure between each.
    Figure Legend Snippet: Single and double glycosylation mutants of β2 can promote surface localization of NaV1.5. MDCK cells (A, C, and E) stably expressing the indicated single mutant for β2-YFP glycosylation were transiently transfected with the vector SCN5A-FLAG and grown polarized in Transwells. Cells were fixed and immunostained with a rabbit polyclonal antibody against NaV1.5 (red), and with a mouse mAb to gp114 (cyan). Images were obtained by confocal microscopy. In merged images, the YFP-emitted fluorescence is in green, and DAPI is in gray. Representative xy sections taken at the apical level (section level chosen to assess presence of NaV1.5 at the apical surface) and corresponding z axis reconstruction (reciprocal xz and xy sections marked by a yellow dashed line) show noticeable apical localization of NaV1.5 with the different β2 variants. Scale bars, 10 μm. B, D, and F, line charts displaying the CTCF (mean percentage ± S.D. (error bars)) along an apical-to-basal z-stack (section 1: most apical; 0.5-μm optical slice thickness) show the NaV1.5 curve peak in close proximity to those of apical gp114 and any of the β2 mutants. DAPI is included as reference for the nuclear level (≥6 cells were analyzed per condition). G and I, MDCK cells stably expressing NaV1.5-YFP were transiently cotransfected with the SCN2B-yfp vector to express β2-YFP, WT, or any of the indicated single (G) or double (I) mutants, plus additional SCN5A-FLAG vector to ensure extensive NaV1.5 overexpression, and grown overnight in wells. Cells were surface-biotinylated at 4 °C. The same amount of protein was used to process each lysate (∼600 μg), 97% of which was subjected to overnight NeutrAvidin pulldown. Representative Western blots (G and I) and band quantitation (H and J) show comparable levels of NaV1.5 in biotin-NeutrAvidin pulldowns (Membrane) in the presence of any mutant variant of β2 as with the WT. One-way ANOVA revealed no differences among means. Data are mean ± S.D. (n ≥ 3). Na/K-ATPase was blotted as surface marker to correct for quantitations in pulldowns. Molecular mass markers are in kDa. For clear display, the blots in G and I show lysates and pulldowns separated by division lines, which indicate different exposure between each.

    Techniques Used: Stable Transfection, Expressing, Mutagenesis, Transfection, Plasmid Preparation, Confocal Microscopy, Fluorescence, Over Expression, Western Blot, Quantitation Assay, Variant Assay, Marker

    polyclonal rabbit antibody  (Alomone Labs)


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    Alomone Labs polyclonal rabbit antibody
    Polyclonal Rabbit Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/polyclonal rabbit antibody/product/Alomone Labs
    Average 91 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    polyclonal rabbit antibody - by Bioz Stars, 2023-01
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    Alomone Labs rabbit polyclonal antibodies
    Unglycosylated β2 is retained in the ER. MDCK cells were transiently transfected with the SCN2B-yfp vector to express WT or fully unglycosylated β2 (ung) and grown for 1 day in wells. Cells were treated with TUN 2 h after transfection or left untreated (−), fixed, and immunostained with a rabbit <t>polyclonal</t> antibody against calnexin (red). A, representative xy sections show that unglycosylated β2 (green) is intracellular and overlaps with calnexin, as does the WT in TUN-treated cells. This contrasts with the localization of β2 WT at the cell end in untreated cells, also displaying a scattered pattern. To focus more accurately where β2 is found in each condition, sections were taken right above the nucleus (WT −) or at the nuclear level (for the rest). Nuclear staining by DAPI is in blue. Scale bar, 10 μm. B, line chart showing Manders' coefficients calculated along the z axis and indicating the fraction of β2 overlapping to compartments labeled with calnexin. The high overlap in TUN-treated cells and in those expressing unglycosylated β2 contrasts with negligible overlap in untreated cells expressing β2 WT. One-way ANOVA with Tukey's HSD post hoc test revealed differences among means (*, p < 0.0005). Data are mean ± S.D. (error bars) (n ≥ 3).
    Rabbit Polyclonal Antibodies, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit polyclonal antibodies/product/Alomone Labs
    Average 91 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit polyclonal antibodies - by Bioz Stars, 2023-01
    91/100 stars
      Buy from Supplier

    91
    Alomone Labs polyclonal rabbit antibody
    Unglycosylated β2 is retained in the ER. MDCK cells were transiently transfected with the SCN2B-yfp vector to express WT or fully unglycosylated β2 (ung) and grown for 1 day in wells. Cells were treated with TUN 2 h after transfection or left untreated (−), fixed, and immunostained with a rabbit <t>polyclonal</t> antibody against calnexin (red). A, representative xy sections show that unglycosylated β2 (green) is intracellular and overlaps with calnexin, as does the WT in TUN-treated cells. This contrasts with the localization of β2 WT at the cell end in untreated cells, also displaying a scattered pattern. To focus more accurately where β2 is found in each condition, sections were taken right above the nucleus (WT −) or at the nuclear level (for the rest). Nuclear staining by DAPI is in blue. Scale bar, 10 μm. B, line chart showing Manders' coefficients calculated along the z axis and indicating the fraction of β2 overlapping to compartments labeled with calnexin. The high overlap in TUN-treated cells and in those expressing unglycosylated β2 contrasts with negligible overlap in untreated cells expressing β2 WT. One-way ANOVA with Tukey's HSD post hoc test revealed differences among means (*, p < 0.0005). Data are mean ± S.D. (error bars) (n ≥ 3).
    Polyclonal Rabbit Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/polyclonal rabbit antibody/product/Alomone Labs
    Average 91 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    polyclonal rabbit antibody - by Bioz Stars, 2023-01
    91/100 stars
      Buy from Supplier

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    Unglycosylated β2 is retained in the ER. MDCK cells were transiently transfected with the SCN2B-yfp vector to express WT or fully unglycosylated β2 (ung) and grown for 1 day in wells. Cells were treated with TUN 2 h after transfection or left untreated (−), fixed, and immunostained with a rabbit polyclonal antibody against calnexin (red). A, representative xy sections show that unglycosylated β2 (green) is intracellular and overlaps with calnexin, as does the WT in TUN-treated cells. This contrasts with the localization of β2 WT at the cell end in untreated cells, also displaying a scattered pattern. To focus more accurately where β2 is found in each condition, sections were taken right above the nucleus (WT −) or at the nuclear level (for the rest). Nuclear staining by DAPI is in blue. Scale bar, 10 μm. B, line chart showing Manders' coefficients calculated along the z axis and indicating the fraction of β2 overlapping to compartments labeled with calnexin. The high overlap in TUN-treated cells and in those expressing unglycosylated β2 contrasts with negligible overlap in untreated cells expressing β2 WT. One-way ANOVA with Tukey's HSD post hoc test revealed differences among means (*, p < 0.0005). Data are mean ± S.D. (error bars) (n ≥ 3).

    Journal: The Journal of Biological Chemistry

    Article Title: N -Glycosylation of the voltage-gated sodium channel β2 subunit is required for efficient trafficking of Na V 1.5/β2 to the plasma membrane

    doi: 10.1074/jbc.RA119.007903

    Figure Lengend Snippet: Unglycosylated β2 is retained in the ER. MDCK cells were transiently transfected with the SCN2B-yfp vector to express WT or fully unglycosylated β2 (ung) and grown for 1 day in wells. Cells were treated with TUN 2 h after transfection or left untreated (−), fixed, and immunostained with a rabbit polyclonal antibody against calnexin (red). A, representative xy sections show that unglycosylated β2 (green) is intracellular and overlaps with calnexin, as does the WT in TUN-treated cells. This contrasts with the localization of β2 WT at the cell end in untreated cells, also displaying a scattered pattern. To focus more accurately where β2 is found in each condition, sections were taken right above the nucleus (WT −) or at the nuclear level (for the rest). Nuclear staining by DAPI is in blue. Scale bar, 10 μm. B, line chart showing Manders' coefficients calculated along the z axis and indicating the fraction of β2 overlapping to compartments labeled with calnexin. The high overlap in TUN-treated cells and in those expressing unglycosylated β2 contrasts with negligible overlap in untreated cells expressing β2 WT. One-way ANOVA with Tukey's HSD post hoc test revealed differences among means (*, p < 0.0005). Data are mean ± S.D. (error bars) (n ≥ 3).

    Article Snippet: Commercial rabbit polyclonal antibodies used were from Alomone (ASC-013 to Na V 1.5 and ASC-007 to anti-β2), from Abcam (anti-GFP (ab290) and anti-calnexin (ab75801)), and from Sigma (anti-actin (A 2066)).

    Techniques: Transfection, Plasmid Preparation, Staining, Labeling, Expressing

    BFA prevents complex glycosylation of β2, a fraction of which can reach the cell surface. MDCK cells were transiently transfected with the SCN2B-yfp vector to express WT or fully unglycosylated β2 (ung) and then treated 2 h later with BFA (+) or left untreated (−) and grown overnight in wells. A and C, cells were surface-biotinylated at 4 °C. The same amount of protein was used to process each lysate (∼100 μg), and the corresponding portion (nine-tenths) was subjected to overnight pulldown. Denatured protein from cell lysates and pulldowns was treated overnight at 37 °C with Endo H to cleave off immature N-glycans or left untreated (−). Representative Western blots show that the (lower) faster-migrating band of β2 WT is the only one visible in cells treated with BFA and increases its mobility with the Endo H treatment; this band coincides with unglycosylated β2 (C; compare with Fig. 2A). Note that Endo H digestion in pulldowns is only partial, either due to saturation of the enzyme or to suboptimal conditions for enzyme action. Blots for Na/K-ATPase are included as loading controls. Molecular mass markers are in kDa. B, band quantitation shows reduced levels of immature β2 in biotin-NeutrAvidin pulldowns (Membrane) of BFA-treated cells. Two-tailed Student's t test shows significant difference (*, p < 0.05). Data are mean ± S.D. (error bars) (n ≥ 3). D, cells were fixed and immunostained with a rabbit polyclonal antibody against calnexin (red) and a mouse monoclonal to GM130 (blue). Representative xy sections show that, in BFA-treated cells, β2 WT displays an intracellular accumulation comparable with mutated β2 (green), grossly overlapping with calnexin in enlarged structures (arrowheads). This contrasts with its apparent localization in the plasma membrane in untreated cells, displaying also a scattered pattern that does not overlap with calnexin (sections were taken at the cell level where β2 is mainly found in each case). Nuclear staining by DAPI is shown in gray. Scale bar, 10 μm.

    Journal: The Journal of Biological Chemistry

    Article Title: N -Glycosylation of the voltage-gated sodium channel β2 subunit is required for efficient trafficking of Na V 1.5/β2 to the plasma membrane

    doi: 10.1074/jbc.RA119.007903

    Figure Lengend Snippet: BFA prevents complex glycosylation of β2, a fraction of which can reach the cell surface. MDCK cells were transiently transfected with the SCN2B-yfp vector to express WT or fully unglycosylated β2 (ung) and then treated 2 h later with BFA (+) or left untreated (−) and grown overnight in wells. A and C, cells were surface-biotinylated at 4 °C. The same amount of protein was used to process each lysate (∼100 μg), and the corresponding portion (nine-tenths) was subjected to overnight pulldown. Denatured protein from cell lysates and pulldowns was treated overnight at 37 °C with Endo H to cleave off immature N-glycans or left untreated (−). Representative Western blots show that the (lower) faster-migrating band of β2 WT is the only one visible in cells treated with BFA and increases its mobility with the Endo H treatment; this band coincides with unglycosylated β2 (C; compare with Fig. 2A). Note that Endo H digestion in pulldowns is only partial, either due to saturation of the enzyme or to suboptimal conditions for enzyme action. Blots for Na/K-ATPase are included as loading controls. Molecular mass markers are in kDa. B, band quantitation shows reduced levels of immature β2 in biotin-NeutrAvidin pulldowns (Membrane) of BFA-treated cells. Two-tailed Student's t test shows significant difference (*, p < 0.05). Data are mean ± S.D. (error bars) (n ≥ 3). D, cells were fixed and immunostained with a rabbit polyclonal antibody against calnexin (red) and a mouse monoclonal to GM130 (blue). Representative xy sections show that, in BFA-treated cells, β2 WT displays an intracellular accumulation comparable with mutated β2 (green), grossly overlapping with calnexin in enlarged structures (arrowheads). This contrasts with its apparent localization in the plasma membrane in untreated cells, displaying also a scattered pattern that does not overlap with calnexin (sections were taken at the cell level where β2 is mainly found in each case). Nuclear staining by DAPI is shown in gray. Scale bar, 10 μm.

    Article Snippet: Commercial rabbit polyclonal antibodies used were from Alomone (ASC-013 to Na V 1.5 and ASC-007 to anti-β2), from Abcam (anti-GFP (ab290) and anti-calnexin (ab75801)), and from Sigma (anti-actin (A 2066)).

    Techniques: Transfection, Plasmid Preparation, Western Blot, Quantitation Assay, Two Tailed Test, Staining

    Surface localization of NaV1.5 is reduced with unglycosylated β2. A and B, MDCK cells stably expressing WT or fully unglycosylated (ung) β2-YFP were transiently transfected with the vector SCN5A-FLAG and grown polarized in Transwells. Cells were fixed and immunostained with a rabbit polyclonal antibody against NaV1.5 (red) and with a mouse mAb to gp114 (cyan). Images were obtained by confocal microscopy. In merged images, the YFP-emitted fluorescence is shown in green and DAPI is in blue. Representative xy sections taken at the apical (A) or nuclear (B) levels (sections taken at the cell level where NaV1.5 is mainly found in each case) and corresponding z axis reconstruction (reciprocal xz and xy sections marked by a yellow dashed line) show improved apical localization of NaV1.5 with β2 WT (A), which remains mostly intracellular in the presence of unglycosylated β2 (B); note the intracellular NaV1.5 accumulation with mutated β2 (arrowhead). Scale bars, 10 μm. C and D, line charts displaying the CTCF (mean percentage ± S.D. (error bars)) along an apical-to-basal z-stack (section 1: most apical; 0.5-μm optical slice thickness) show the NaV1.5 curve peak close to those of apical gp114 and β2 WT (C). In contrast, NaV1.5 is displaced toward the nuclear section with mutated β2, which overlays with DAPI (D), included as reference for the nuclear level (≥6 cells were analyzed per condition). E, MDCK cells stably expressing NaV1.5-YFP were transiently cotransfected with the SCN2B-yfp vector to express β2, WT or fully unglycosylated (ung), plus additional SCN5A-FLAG vector to ensure extensive NaV1.5 overexpression, and grown overnight in wells; the pEGFP-N1 vector was used as a control. Cells were surface-biotinylated at 4 °C. The same amount of protein was used to process each lysate (∼600 μg), 97% of which was subjected to overnight NeutrAvidin pulldown. Representative Western blots and band quantitation (F) show reduced levels of NaV1.5 in biotin-NeutrAvidin pulldowns (Membrane) in the presence of unglycosylated β2 or without β2 (GFP), when comparing with the WT. One-way ANOVA with Tukey's HSD post hoc test showed significant differences (*, p < 0.002). The percentage of NaV1.5 at the cell surface over total cellular NaV1.5 protein varied from 1.42 ± 0.98 in the WT to 0.73 ± 0.50% with unglycosylated β2. Data are mean ± S.D. (n ≥ 6). Na/K-ATPase was blotted as surface marker to correct for quantitations in pulldowns. Molecular mass markers are in kDa. For clear display, the blot in E shows lysates and pulldowns separated by division lines, which indicate different exposure between lysates and pulldowns but equal exposure within each group.

    Journal: The Journal of Biological Chemistry

    Article Title: N -Glycosylation of the voltage-gated sodium channel β2 subunit is required for efficient trafficking of Na V 1.5/β2 to the plasma membrane

    doi: 10.1074/jbc.RA119.007903

    Figure Lengend Snippet: Surface localization of NaV1.5 is reduced with unglycosylated β2. A and B, MDCK cells stably expressing WT or fully unglycosylated (ung) β2-YFP were transiently transfected with the vector SCN5A-FLAG and grown polarized in Transwells. Cells were fixed and immunostained with a rabbit polyclonal antibody against NaV1.5 (red) and with a mouse mAb to gp114 (cyan). Images were obtained by confocal microscopy. In merged images, the YFP-emitted fluorescence is shown in green and DAPI is in blue. Representative xy sections taken at the apical (A) or nuclear (B) levels (sections taken at the cell level where NaV1.5 is mainly found in each case) and corresponding z axis reconstruction (reciprocal xz and xy sections marked by a yellow dashed line) show improved apical localization of NaV1.5 with β2 WT (A), which remains mostly intracellular in the presence of unglycosylated β2 (B); note the intracellular NaV1.5 accumulation with mutated β2 (arrowhead). Scale bars, 10 μm. C and D, line charts displaying the CTCF (mean percentage ± S.D. (error bars)) along an apical-to-basal z-stack (section 1: most apical; 0.5-μm optical slice thickness) show the NaV1.5 curve peak close to those of apical gp114 and β2 WT (C). In contrast, NaV1.5 is displaced toward the nuclear section with mutated β2, which overlays with DAPI (D), included as reference for the nuclear level (≥6 cells were analyzed per condition). E, MDCK cells stably expressing NaV1.5-YFP were transiently cotransfected with the SCN2B-yfp vector to express β2, WT or fully unglycosylated (ung), plus additional SCN5A-FLAG vector to ensure extensive NaV1.5 overexpression, and grown overnight in wells; the pEGFP-N1 vector was used as a control. Cells were surface-biotinylated at 4 °C. The same amount of protein was used to process each lysate (∼600 μg), 97% of which was subjected to overnight NeutrAvidin pulldown. Representative Western blots and band quantitation (F) show reduced levels of NaV1.5 in biotin-NeutrAvidin pulldowns (Membrane) in the presence of unglycosylated β2 or without β2 (GFP), when comparing with the WT. One-way ANOVA with Tukey's HSD post hoc test showed significant differences (*, p < 0.002). The percentage of NaV1.5 at the cell surface over total cellular NaV1.5 protein varied from 1.42 ± 0.98 in the WT to 0.73 ± 0.50% with unglycosylated β2. Data are mean ± S.D. (n ≥ 6). Na/K-ATPase was blotted as surface marker to correct for quantitations in pulldowns. Molecular mass markers are in kDa. For clear display, the blot in E shows lysates and pulldowns separated by division lines, which indicate different exposure between lysates and pulldowns but equal exposure within each group.

    Article Snippet: Commercial rabbit polyclonal antibodies used were from Alomone (ASC-013 to Na V 1.5 and ASC-007 to anti-β2), from Abcam (anti-GFP (ab290) and anti-calnexin (ab75801)), and from Sigma (anti-actin (A 2066)).

    Techniques: Stable Transfection, Expressing, Transfection, Plasmid Preparation, Confocal Microscopy, Fluorescence, Over Expression, Western Blot, Quantitation Assay, Marker

    Defect over time of unglycosylated β2 in promoting surface localization of NaV1.5. MDCK cells stably expressing WT (A) or fully unglycosylated (ung) β2-YFP (C) or untransfected (parental) cells (E) were transiently transfected with the vector SCN5A-FLAG and grown in wells for the indicated number of days. Cells were fixed and immunostained with a rabbit polyclonal antibody against NaV1.5 (red) and with a mouse mAb to Na/K-ATPase (blue). Images were obtained by confocal microscopy. In merged images, the YFP-emitted fluorescence is shown in green and DAPI is in gray. A, C, and E, representative xy sections (sections taken at the cell level where NaV1.5 is mainly found in each case) show a general diffuse NaV1.5 pattern, intracellular and often perinuclear, except for a noticeable overlap with Na/K-ATPase, particularly at day 1, in the presence of β2 WT. Scale bars, 10 μm. Confocal images were analyzed by calculating the MFI along linear segments of 30 pixels in length (d, distance; 0.1 μm/pixel) drawn from the cell end perpendicularly into the cytoplasm. B, D, and F, line charts show MFIs with the first 5 pixels of the segments, equivalent to the plasma membrane region (cell end), marked with a square bracket. The highest MFI levels are at the cell end for Na/K-ATPase and for β2 WT, which progressively decrease intracellularly. The profile for NaV1.5 increases at the cell end only in the presence of β2 WT and especially at day 1 (B) but remains comparatively low within this region with unglycosylated β2 (D) or in the absence of β2 (F). Data are mean ± S.D. (error bars) (number of cells analyzed ≥3; 4 segments/cell).

    Journal: The Journal of Biological Chemistry

    Article Title: N -Glycosylation of the voltage-gated sodium channel β2 subunit is required for efficient trafficking of Na V 1.5/β2 to the plasma membrane

    doi: 10.1074/jbc.RA119.007903

    Figure Lengend Snippet: Defect over time of unglycosylated β2 in promoting surface localization of NaV1.5. MDCK cells stably expressing WT (A) or fully unglycosylated (ung) β2-YFP (C) or untransfected (parental) cells (E) were transiently transfected with the vector SCN5A-FLAG and grown in wells for the indicated number of days. Cells were fixed and immunostained with a rabbit polyclonal antibody against NaV1.5 (red) and with a mouse mAb to Na/K-ATPase (blue). Images were obtained by confocal microscopy. In merged images, the YFP-emitted fluorescence is shown in green and DAPI is in gray. A, C, and E, representative xy sections (sections taken at the cell level where NaV1.5 is mainly found in each case) show a general diffuse NaV1.5 pattern, intracellular and often perinuclear, except for a noticeable overlap with Na/K-ATPase, particularly at day 1, in the presence of β2 WT. Scale bars, 10 μm. Confocal images were analyzed by calculating the MFI along linear segments of 30 pixels in length (d, distance; 0.1 μm/pixel) drawn from the cell end perpendicularly into the cytoplasm. B, D, and F, line charts show MFIs with the first 5 pixels of the segments, equivalent to the plasma membrane region (cell end), marked with a square bracket. The highest MFI levels are at the cell end for Na/K-ATPase and for β2 WT, which progressively decrease intracellularly. The profile for NaV1.5 increases at the cell end only in the presence of β2 WT and especially at day 1 (B) but remains comparatively low within this region with unglycosylated β2 (D) or in the absence of β2 (F). Data are mean ± S.D. (error bars) (number of cells analyzed ≥3; 4 segments/cell).

    Article Snippet: Commercial rabbit polyclonal antibodies used were from Alomone (ASC-013 to Na V 1.5 and ASC-007 to anti-β2), from Abcam (anti-GFP (ab290) and anti-calnexin (ab75801)), and from Sigma (anti-actin (A 2066)).

    Techniques: Stable Transfection, Expressing, Transfection, Plasmid Preparation, Confocal Microscopy, Fluorescence

    Single β2 glycosylation mutants can promote surface localization of NaV1.5; analysis over time. MDCK cells stably expressing the indicated single mutant for β2-YFP glycosylation were transiently transfected with the vector SCN5A-FLAG and grown in wells for the specified number of days. Cells were fixed and immunostained with a rabbit polyclonal antibody against NaV1.5 (red) and with a mouse mAb to Na/K-ATPase (blue). Images were obtained by confocal microscopy. In merged images, the YFP-emitted fluorescence is in green and DAPI is in gray. A, C, and E, Representative xy sections (taken at the level where NaV1.5 is mainly found in each case) show some areas of overlap of NaV1.5 with Na/K-ATPase at the cell end, particularly at day 1, in the presence of any of the mutants, while remaining mostly disperse throughout the cell at later time points. Scale bars, 10 μm. Confocal images were analyzed by calculating the MFI along linear segments of 30 pixels in length (d, distance; 0.1 μm/pixel) drawn from the cell end perpendicularly into the cytoplasm. B, D, and F, line charts show MFIs with the first 5 pixels of the segments, equivalent to the plasma membrane region (cell end), marked with a square bracket. The highest MFI levels are at the cell end for Na/K-ATPase and for the different β2 single mutants, all progressively decreasing intracellularly. The profile for NaV1.5 increases at the cell end at day 1 in all cases, remaining comparatively low within this region at later time points. Data are mean ± S.D. (error bars) (number of cells analyzed ≥3, 4 segments/cell).

    Journal: The Journal of Biological Chemistry

    Article Title: N -Glycosylation of the voltage-gated sodium channel β2 subunit is required for efficient trafficking of Na V 1.5/β2 to the plasma membrane

    doi: 10.1074/jbc.RA119.007903

    Figure Lengend Snippet: Single β2 glycosylation mutants can promote surface localization of NaV1.5; analysis over time. MDCK cells stably expressing the indicated single mutant for β2-YFP glycosylation were transiently transfected with the vector SCN5A-FLAG and grown in wells for the specified number of days. Cells were fixed and immunostained with a rabbit polyclonal antibody against NaV1.5 (red) and with a mouse mAb to Na/K-ATPase (blue). Images were obtained by confocal microscopy. In merged images, the YFP-emitted fluorescence is in green and DAPI is in gray. A, C, and E, Representative xy sections (taken at the level where NaV1.5 is mainly found in each case) show some areas of overlap of NaV1.5 with Na/K-ATPase at the cell end, particularly at day 1, in the presence of any of the mutants, while remaining mostly disperse throughout the cell at later time points. Scale bars, 10 μm. Confocal images were analyzed by calculating the MFI along linear segments of 30 pixels in length (d, distance; 0.1 μm/pixel) drawn from the cell end perpendicularly into the cytoplasm. B, D, and F, line charts show MFIs with the first 5 pixels of the segments, equivalent to the plasma membrane region (cell end), marked with a square bracket. The highest MFI levels are at the cell end for Na/K-ATPase and for the different β2 single mutants, all progressively decreasing intracellularly. The profile for NaV1.5 increases at the cell end at day 1 in all cases, remaining comparatively low within this region at later time points. Data are mean ± S.D. (error bars) (number of cells analyzed ≥3, 4 segments/cell).

    Article Snippet: Commercial rabbit polyclonal antibodies used were from Alomone (ASC-013 to Na V 1.5 and ASC-007 to anti-β2), from Abcam (anti-GFP (ab290) and anti-calnexin (ab75801)), and from Sigma (anti-actin (A 2066)).

    Techniques: Stable Transfection, Expressing, Mutagenesis, Transfection, Plasmid Preparation, Confocal Microscopy, Fluorescence

    Single and double glycosylation mutants of β2 can promote surface localization of NaV1.5. MDCK cells (A, C, and E) stably expressing the indicated single mutant for β2-YFP glycosylation were transiently transfected with the vector SCN5A-FLAG and grown polarized in Transwells. Cells were fixed and immunostained with a rabbit polyclonal antibody against NaV1.5 (red), and with a mouse mAb to gp114 (cyan). Images were obtained by confocal microscopy. In merged images, the YFP-emitted fluorescence is in green, and DAPI is in gray. Representative xy sections taken at the apical level (section level chosen to assess presence of NaV1.5 at the apical surface) and corresponding z axis reconstruction (reciprocal xz and xy sections marked by a yellow dashed line) show noticeable apical localization of NaV1.5 with the different β2 variants. Scale bars, 10 μm. B, D, and F, line charts displaying the CTCF (mean percentage ± S.D. (error bars)) along an apical-to-basal z-stack (section 1: most apical; 0.5-μm optical slice thickness) show the NaV1.5 curve peak in close proximity to those of apical gp114 and any of the β2 mutants. DAPI is included as reference for the nuclear level (≥6 cells were analyzed per condition). G and I, MDCK cells stably expressing NaV1.5-YFP were transiently cotransfected with the SCN2B-yfp vector to express β2-YFP, WT, or any of the indicated single (G) or double (I) mutants, plus additional SCN5A-FLAG vector to ensure extensive NaV1.5 overexpression, and grown overnight in wells. Cells were surface-biotinylated at 4 °C. The same amount of protein was used to process each lysate (∼600 μg), 97% of which was subjected to overnight NeutrAvidin pulldown. Representative Western blots (G and I) and band quantitation (H and J) show comparable levels of NaV1.5 in biotin-NeutrAvidin pulldowns (Membrane) in the presence of any mutant variant of β2 as with the WT. One-way ANOVA revealed no differences among means. Data are mean ± S.D. (n ≥ 3). Na/K-ATPase was blotted as surface marker to correct for quantitations in pulldowns. Molecular mass markers are in kDa. For clear display, the blots in G and I show lysates and pulldowns separated by division lines, which indicate different exposure between each.

    Journal: The Journal of Biological Chemistry

    Article Title: N -Glycosylation of the voltage-gated sodium channel β2 subunit is required for efficient trafficking of Na V 1.5/β2 to the plasma membrane

    doi: 10.1074/jbc.RA119.007903

    Figure Lengend Snippet: Single and double glycosylation mutants of β2 can promote surface localization of NaV1.5. MDCK cells (A, C, and E) stably expressing the indicated single mutant for β2-YFP glycosylation were transiently transfected with the vector SCN5A-FLAG and grown polarized in Transwells. Cells were fixed and immunostained with a rabbit polyclonal antibody against NaV1.5 (red), and with a mouse mAb to gp114 (cyan). Images were obtained by confocal microscopy. In merged images, the YFP-emitted fluorescence is in green, and DAPI is in gray. Representative xy sections taken at the apical level (section level chosen to assess presence of NaV1.5 at the apical surface) and corresponding z axis reconstruction (reciprocal xz and xy sections marked by a yellow dashed line) show noticeable apical localization of NaV1.5 with the different β2 variants. Scale bars, 10 μm. B, D, and F, line charts displaying the CTCF (mean percentage ± S.D. (error bars)) along an apical-to-basal z-stack (section 1: most apical; 0.5-μm optical slice thickness) show the NaV1.5 curve peak in close proximity to those of apical gp114 and any of the β2 mutants. DAPI is included as reference for the nuclear level (≥6 cells were analyzed per condition). G and I, MDCK cells stably expressing NaV1.5-YFP were transiently cotransfected with the SCN2B-yfp vector to express β2-YFP, WT, or any of the indicated single (G) or double (I) mutants, plus additional SCN5A-FLAG vector to ensure extensive NaV1.5 overexpression, and grown overnight in wells. Cells were surface-biotinylated at 4 °C. The same amount of protein was used to process each lysate (∼600 μg), 97% of which was subjected to overnight NeutrAvidin pulldown. Representative Western blots (G and I) and band quantitation (H and J) show comparable levels of NaV1.5 in biotin-NeutrAvidin pulldowns (Membrane) in the presence of any mutant variant of β2 as with the WT. One-way ANOVA revealed no differences among means. Data are mean ± S.D. (n ≥ 3). Na/K-ATPase was blotted as surface marker to correct for quantitations in pulldowns. Molecular mass markers are in kDa. For clear display, the blots in G and I show lysates and pulldowns separated by division lines, which indicate different exposure between each.

    Article Snippet: Commercial rabbit polyclonal antibodies used were from Alomone (ASC-013 to Na V 1.5 and ASC-007 to anti-β2), from Abcam (anti-GFP (ab290) and anti-calnexin (ab75801)), and from Sigma (anti-actin (A 2066)).

    Techniques: Stable Transfection, Expressing, Mutagenesis, Transfection, Plasmid Preparation, Confocal Microscopy, Fluorescence, Over Expression, Western Blot, Quantitation Assay, Variant Assay, Marker