Figure 4. Altered subcellular... | Rockefeller University Press

$Figure 4. Altered subcellular localization and rescue of expression of Kv2.1 V378A in COS-1 cells. (A) Transfection efficiency of wild-type, V378A, and an equal amount of wild-type + V378A (Het) in COS-1 cells, as measured by immunofluorescence. Data are from at least 115 cells per independent sample (n = 3 for each treatment). Data are the mean ± SEM. *, P < 0.05 (two-tailed unpaired t test). (B) Quantitation of surface expression of Kv2.1 in the absence or presence of 400 nM GxTX (n = 3 per treatment). (C) Multiplex fluorescence labeling for Kv2.1 (red) and Hoechst 33258 (blue) in cells cultured in the absence or presence of 400 nM GxTX. Wild-type Kv2.1 expression was robust and associated with the plasma membrane regardless of treatment. In contrast, V378A expression was, in a significant fraction of cells, restricted to an intracellular perinuclear compartment consistent with the Golgi apparatus, whereas V378A cells cultured in the presence of 400 nM GxTX had more plasma membrane–associated labeling, resembling that of wild-type Kv2.1. Bar, 10 µm. (D) Multiplex fluorescence labeling of a Kv2.1 V378A-expressing cell for Kv2.1 (red), the Golgi marker Lens culinaris lectin (green), and Hoechst 33258 (blue, to label nuclei) suggests a concentration of Kv2.1 V378A in the Golgi apparatus. Insets are a higher magnification of the perinuclear region. Bar, 10 µm. (E) Immunoblot analysis of COS-1 cell extracts cultured in the absence or presence of 400 nM GxTX, showing reduced levels of V378A mutant Kv2.1 protein that is rescued by GxTX. Mortalin was used as a loading control. (F) Quantitation of Kv2.1 immunoblot expression normalized to the mortalin loading control indicates a significantly decreased level of expression of Kv2.1 V378A relative to wild-type Kv2.1. Culturing cells in the presence of 400 nM GxTX rescues the decreased V378A expression level in this cell type (n = 3 per treatment). *, P < 0.05, by two-way ANOVA followed by post-hoc Tukey tests.$

Figure 4.

Altered subcellular localization and rescue of expression of Kv2.1 V378A in COS-1 cells. (A) Transfection efficiency of wild-type, V378A, and an equal amount of wild-type + V378A (Het) in COS-1 cells, as measured by immunofluorescence. Data are from at least 115 cells per independent sample (n = 3 for each treatment). Data are the mean ± SEM. *, P < 0.05 (two-tailed unpaired t test). (B) Quantitation of surface expression of Kv2.1 in the absence or presence of 400 nM GxTX (n = 3 per treatment). (C) Multiplex fluorescence labeling for Kv2.1 (red) and Hoechst 33258 (blue) in cells cultured in the absence or presence of 400 nM GxTX. Wild-type Kv2.1 expression was robust and associated with the plasma membrane regardless of treatment. In contrast, V378A expression was, in a significant fraction of cells, restricted to an intracellular perinuclear compartment consistent with the Golgi apparatus, whereas V378A cells cultured in the presence of 400 nM GxTX had more plasma membrane–associated labeling, resembling that of wild-type Kv2.1. Bar, 10 µm. (D) Multiplex fluorescence labeling of a Kv2.1 V378A-expressing cell for Kv2.1 (red), the Golgi marker Lens culinaris lectin (green), and Hoechst 33258 (blue, to label nuclei) suggests a concentration of Kv2.1 V378A in the Golgi apparatus. Insets are a higher magnification of the perinuclear region. Bar, 10 µm. (E) Immunoblot analysis of COS-1 cell extracts cultured in the absence or presence of 400 nM GxTX, showing reduced levels of V378A mutant Kv2.1 protein that is rescued by GxTX. Mortalin was used as a loading control. (F) Quantitation of Kv2.1 immunoblot expression normalized to the mortalin loading control indicates a significantly decreased level of expression of Kv2.1 V378A relative to wild-type Kv2.1. Culturing cells in the presence of 400 nM GxTX rescues the decreased V378A expression level in this cell type (n = 3 per treatment). *, P < 0.05, by two-way ANOVA followed by post-hoc Tukey tests.

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