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. 2010 Apr 1;285(23):17938–17953. doi: 10.1074/jbc.M109.086553

FIGURE 6.

FIGURE 6.

DN Rab35 co-localizes with KCa2.3 and prevents recycling to the plasma membrane. BLAP-tagged KCa2.3 was co-expressed with either GFP-tagged Rab35 (A) or DN Rab35 (B) in HEK cells; the plasma membrane channel was labeled with streptavidin-Alexa555, and localization was determined following incubation at 37 °C for the times indicated. A, in the presence of Rab35, KCa2.3 was localized primarily at the plasma membrane after 5 h at 37 °C, identical to what was observed when KCa2.3 is expressed alone. B, at time 0 h, KCa2.3 is localized to the plasma membrane (top panels). However, after 5 h at 37 °C, KCa2.3 localizes to the DN Rab35-induced intracellular vacuoles. C, BLAP-tagged KCa2.3 was co-expressed with GFP-tagged DN Rab35 in HMEC-1 cells, and the plasma membrane channel was labeled with streptavidin-Alexa555, and localization was determined following incubation at 37 °C for 5 h. KCa2.3 localizes with DN Rab35 after 5 h and is nearly absent from the membrane demonstrating that recycling is impaired. Nuclei are labeled with DAPI (blue). Projection images from multiple z-sections are shown. D, immunoblot of total lysate following co-expression of KCa2.3 with WT Rab35 (1st lane) or DN Rab35 (2nd lane). Rab35 expression had no effect on expression of KCa2.3. E, cell surface KCa2.3 expression was evaluated by biotinylation following co-expression with either Rab35 (1st lane) or DN Rab35 (2nd lane). DN Rab35 caused a significant decrease (75 ± 9%, n = 3; p < 0.01) in steady-state cell surface expression of KCa2.3. F, KCa2.3 was co-expressed with either WT (left panel) or DN (right panel) Rab35, and cell surface channel was biotinylated. At the indicated times, cells were lysed, subjected to streptavidin pulldown, and blotted for KCa2.3. The bands were quantified by densitometry, normalized, and plotted as a function of time (bottom panel) for DN (solid line, triangles) and WT (dashed line, circles) Rab35. G, co-immunoprecipitation of KCa2.3 with HA-tagged Rab35 was carried out in HEK cells. Rab35 was immunoprecipitated via an HA tag, and the subsequent blot was probed for KCa2.3. Cells expressed either Rab35 plus empty vector (1st lane), KCa2.3 plus vector (2nd lane), or KCa2.3 plus HA-Rab35 (3rd and 4th lanes). The V5 Ab was used as an IgG control in 3rd lane.