FIG. 4.
hERG1A and hERG1B coassemble with hERG1USO and hERG1BUSO. (A) (Right) Coimmunoprecipitation of hERG1USO with hERG1A and hERG1B. Total lysates (1 mg) from HEK-hERG1USO cells transfected with herg1a (HEK-hERG1USO+herg1a) or herg1b (HEK-hERG1USO+herg1b) and HEK 293 cells transiently transfected with herg1a (HEK-herg1a) and herg1b (HEK-herg1b) were immunoprecipitated with anti-USO antibody and run through a 7.5% SDS-polyacrylamide gel, and the following WB was revealed with anti pan-hERG1 antibody. Total lysates (5 μg each) from mock-transfected HEK 293 cells (HEK-MOCK), cells cotransfected with herg1a and herg1b (HEK-herg1a+1b) and from HEK-hERG1USO cells (HEK-hERG1USO) were run. The no-IP lane contains samples in which no antibody was added to cell lysates during immunoprecipitation. (Left) Coimmunoprecipitation of hERG1BUSO with hERG1A and hERG1B. Total lysates (1 mg) from HEK-hERG1BUSO transfected with herg1b (HEK-hERG1BUSO+herg1b), herg1a (HEK-hERG1BUSO+herg1a), and HEK 293 cells transiently transfected with herg1a (HEK-herg1a) and herg1b (HEK-herg1b), were treated as described above for the right gel in panel A. Total lysates (5 μg each) from HEK-hERG1BUSO cells and from HEK 293 cells cotransfected with herg1a and herg1b (HEK-herg1a+1b), were also run. Lane no-IP, no antibody was added during immunoprecipitation. The positions of molecular mass markers (in kilodaltons) are indicated to the left of the gels. (B) Cellular localization of hERG1 in cotransfected cells. HEK-hERG1USO cells were transiently transfected with herg1a and stained with anti-pan-hERG1 (a) or anti-KDEL (b) antibodies. The merged image of panels a and b is shown in panel c. (d) hERG1A/KDEL colocalization, determined through the calculation of the Manders' coefficient (see Materials and Methods). To do this, images from HEK-hERG1USO cells cotransfected with herg1a (from the same dish as the pictures in panels a to c), as well as the images as those reported in Fig. 2F, panel b, were used. The values calculated by the Manders' coefficient are as follows: 0.17 ± 0.01 for 10 cells transfected with herg1a and 0.36 ± 0.06 for 10 HEK-hERG1USO cells cotransfected with herg1a (P < 0.01, Student's t test). (C) Cellular localization of hERG1 in cotransfected cells. HEK-hERG1USO cells, transiently transfected with herg1a, were stained with anti-pan-hERG1 (a) or anti-β1 integrin (b) antibodies. The merged image of panels a and b is shown in panel c. HEK-hERG1USO cells transiently transfected with herg1a and stained with anti-USO (d) and pan-hERG1 (e) antibodies. To quantify the level of hERG1A expression on the plasma membranes of hERG1USO cells cotransfected with herg1a, a sequential labeling protocol was used, after optimizing the blocking step and diluting the secondary antibodies to avoid any aspecific signal. hERG1A protein expression on the plasma membrane was quantified according to reference 13 (see Materials and Methods). Bar, 10 μm (applies to all panels). (f) Distribution of hERG1 intensity in HEK-hERG1USO cells, displaying high and low USO expression. ImageJ and statistical analyses were performed as described in Materials and Methods. Statistical significance for the Mann-Whitney test (two-tailed) is P < 0.0001. The sum of ranks and median value for high-USO-expressing cells are 45 and 1,557, respectively, and the sum of ranks and median value for low-USO-expressing cells are 108 and 822, respectively (n = 20).