Figure 3.
Association and plasma membrane targeting of Kv1.3 in the presence of KCNE4 are not affected by the 145D/E polymorphism. HEK293 cells were transfected with Kv1.3 and different KCNE4 variants (145D, 145E, and 145A). (A) Representative coIP experiments. Total lysates were IP against CFP (KCNE4) and IB against Kv1.3 and KCNE4 (CFP). SM, starting material; IP-, absence of antibody. (B) Quantification of Kv1.3-KCNE4 coIP results from four independent experiments. The values represent the mean ± SE. Statistical analysis: RM one-way ANOVA. (C–E) HEK293 cells were transfected with Kv1.3-HA with or without KCNE4-CFP variants. Nonpermeabilized cells were incubated with an anti-HA antibody and a secondary antibody conjugated to Cy5. The Cy5 intensity was measured by flow cytometry. (C) Plot of HEK293 cells expressing Kv1.3-HA and CFP as a negative control (top), Kv1.3 + KCNE4 145D (center), and Kv1.3 + KCNE4 145E (bottom) with Cy5 (y axis) and CFP (x axis) fluorescence intensity for each combination (left). On the right, histogram for Cy5 intensity of the CFP-positive cells for each condition (circled in red in the left). (D) Schematic representation of cells expressing Kv1.3-HA (black) without (on the left) or with KCNE4 (blue barrel, on the right) with Cy5 (red circle) conjugated to Kv1.3 at the plasma membrane. (E) Quantification of plasma membrane Kv1.3 (PM Kv1.3) normalized to Cy5 intensity. The values represent the mean ± SE of at least 1,500 cells for each condition. Statistical analysis: Brown–Forsythe ANOVA test. Specific P values were as follows: **P < 0.01 (0.0016 vs. +145D; 0.0017 vs. +145E). IP, immunoprecipitated; IB, immunoblotted. Source data are available for this figure: SourceData F3. Refer to the image caption for details. Panel A shows two sets of immunoblot images. The first set displays bands for Kv1.3 and KCNE4 in starting material (SM) for three different KCNE4 variants (145D, 145E, and 145A). The second set shows bands for Kv1.3 and KCNE4 after immunoprecipitation (IP) against CFP (KCNE4). Panel B is a bar graph comparing the ratio of Kv1.3 to KCNE4 for the three variants. The y-axis represents the Kv1.3/KCNE4 ratio in arbitrary units (AU), and the x-axis lists the Kv1.3 variants (plus 145D, plus 145E, plus 145A). The graph indicates no significant difference (n.s.) between the variants. Panel C contains scatter plots and histograms. The scatter plots show Cy5 fluorescence intensity (y-axis) versus CFP fluorescence intensity (x-axis) for cells expressing Kv1.3-HA and CFP as a negative control, Kv1.3 with KCNE4 145D, and Kv1.3 with KCNE4 145E. The histograms display the Cy5 intensity distribution for CFP-positive cells. Panel D includes schematic diagrams illustrating the localization of Kv1.3-HA at the plasma membrane without and with KCNE4. Panel E is a bar graph quantifying plasma membrane Kv1.3 (PM Kv1.3) normalized to Cy5 intensity. The y-axis represents the normalized Cy5 intensity, and the x-axis lists the conditions (Kv1.3, plus 145D, plus 145E). The graph shows a significant reduction in PM Kv1.3 for the plus 145D and plus 145E variants compared to Kv1.3 alone.

Association and plasma membrane targeting of Kv1.3 in the presence of KCNE4 are not affected by the 145D/E polymorphism. HEK293 cells were transfected with Kv1.3 and different KCNE4 variants (145D, 145E, and 145A). (A) Representative coIP experiments. Total lysates were IP against CFP (KCNE4) and IB against Kv1.3 and KCNE4 (CFP). SM, starting material; IP-, absence of antibody. (B) Quantification of Kv1.3-KCNE4 coIP results from four independent experiments. The values represent the mean ± SE. Statistical analysis: RM one-way ANOVA. (C–E) HEK293 cells were transfected with Kv1.3-HA with or without KCNE4-CFP variants. Nonpermeabilized cells were incubated with an anti-HA antibody and a secondary antibody conjugated to Cy5. The Cy5 intensity was measured by flow cytometry. (C) Plot of HEK293 cells expressing Kv1.3-HA and CFP as a negative control (top), Kv1.3 + KCNE4 145D (center), and Kv1.3 + KCNE4 145E (bottom) with Cy5 (y axis) and CFP (x axis) fluorescence intensity for each combination (left). On the right, histogram for Cy5 intensity of the CFP-positive cells for each condition (circled in red in the left). (D) Schematic representation of cells expressing Kv1.3-HA (black) without (on the left) or with KCNE4 (blue barrel, on the right) with Cy5 (red circle) conjugated to Kv1.3 at the plasma membrane. (E) Quantification of plasma membrane Kv1.3 (PM Kv1.3) normalized to Cy5 intensity. The values represent the mean ± SE of at least 1,500 cells for each condition. Statistical analysis: Brown–Forsythe ANOVA test. Specific P values were as follows: **P < 0.01 (0.0016 vs. +145D; 0.0017 vs. +145E). IP, immunoprecipitated; IB, immunoblotted. Source data are available for this figure: SourceData F3.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal