Fig. 6

Cellular co-distribution, co-immunoprecipitation and membrane expression of β1-integrin and KCa3.1 channels. a. Representative immunofluorescence images of KCa3.1 and β1-integrin staining performed on ATII cells using anti-KCa3.1, anti-β1-integrin, anti-rabbit 633 (for KCa3.1 detection) and anti-mouse 488 (for β1-integrin detection) antibodies. Color superposition shows similar cellular distribution of KCa3.1 and β1-integrin in ATII cells (merge panel, Scale bars, 10 μm). No or diffuse signal was detected with the Alexa fluor 488 and Alexa fluor 633 coupled secondary antibodies in control experiments (negative controls). b. Representative immunoblots showing β1-integrin and KCa3.1 co-immunoprecipitations. β1-integrin (upper panels, IB: β1-integrin) and KCa3.1 (lower panels, IB: KCa3.1) proteins were revealed with specific antibodies after β1-integrin and KCa3.1 immunoprecipitation with anti-β1-integrin (lane 2 « β1-integrin IP ») or anti-KCa3.1 (lane 3 « KCa3.1 IP ») antibodies in ATII cell extracts. Endogenous expression of β1-integrin and KCa3.1 proteins in ATII cell lysate is also shown in lane 1, « Total Lysate ». Lanes 4 and 5 are negative control assays showing an absence of band in IB (IB β1-integrin and IB KCa3.1) after IP in the absence of lysate (lane 4, « Negative IP Control (no lysate) ») and in the absence of β-integrin and KCa3.1 antibodies (lane 5, « Negative IP control (no antibody) »). c. The level of β1-integrin and KCa3.1 channel expression in membrane fractions were determined by immunoblotting using anti-β1-integrin and anti-KCa3.1 antibodies. A representative immunoblot is shown in the left panel. The band intensities were compared in control condition (no coating, −) and in the presence of a fibronectin (+) matrix (right panel, n = 11). *p < 0.05