Figure 4.

Characterization of the β₁/hERG1 complex in HEK-hERG1 cells. (A) Colocalization of hERG1 and β₁ proteins. Double immunofluorescence was performed on HEK-hERG1 cells seeded onto glass slides coated with 20 μg/ml FN in DMEM + BSA, by using anti-hERG1 antibodies (C54) and a FITC-conjugated secondary antibody (hERG1) and anti-β₁ antibodies (TS2/16) and a RITC-conjugated secondary antibody (β₁). Photographs were taken with a confocal microscope and merged (merge). A quantitative analysis of confocal images, using the MetaMorph Software (integrated morphometry analysis; Universal Imaging) showed that 18% (±2, n = 12) of the hERG1 protein and of β₁ integrin colocalize in HEK-hERG1 cells. (B) CoIP of β₁ and hERG1 proteins depends on β₁ activity. CoIP experiments were performed on cells kept in suspension (lane suspended) or seeded either onto BSA- (lane BSA)- or FN (lane FN)-coated dishes for 30 min, by using anti-β₁ antibodies (TS2/16) to immunoprecipitate and anti-hERG1 antibodies for the Western blot. Reprobing with anti-β₁ antibodies also is reported in the bottom panel. Inset (center), densitometric analysis of the amount of hERG1 protein that coimmunoprecipitates with β₁ after cell adhesion on different substrates. The analysis was performed as described in Materials and Methods; data are the means ± SEM of three separate experiments. ^*, significantly different p = 0.00016, Student's t test (FN adherent cells vs. BSA adherent cells); ^**, significantly different p = 0.0000016, Student's t test (FN adherent cells vs. suspended cells). Inset (right), HEK-hERG1 cells were kept in suspension and treated (lane +TS2/16) or not (lane –TS2/16) for 30 min with activating monoclonal anti-β₁ antibodies as described in Materials and Methods. CoIP experiments were performed as described above. (C and D) Colocalization of β₁ with caveolin-1 and hERG1 proteins. Proteins were extracted from HEK-hERG1 cells kept in suspension (lane suspended) or seeded onto FN (lane FN)-coated dishes for 30 min and immunoprecipitated using anti-β₁ antibodies (TS2/16); no-IPs refer to samples where no primary antibody was added to cell lysates, as described in Materials and Methods. Bands were revealed using anti-caveolin-1 antibodies. Reprobing with anti-β₁ antibodies also is reported at the bottom. Inset, total cell lysate obtained from cells treated as in C probed with anti-caveolin-1 antibodies. (D) CoIP of hERG1, caveolin-1 and β₁ proteins. Proteins were extracted from HEK-hERG1 cells kept in suspension (lane suspended) or seeded onto FN-coated dishes for 30 min (lane FN), and immunoprecipitated using anti-hERG1 antibodies (N135); bands were revealed using anti-caveolin-1 antibodies (top), anti-β₁ antibodies (middle), and anti-hERG1 antibodies (C54) (bottom). All the panels in this figure are representative of at least three different experiments, except for TS2/16 treatment (inset to B, right), which was a single experiment. No-IP, no immunoprecipitation.