Fig. 4

Decreased mRNA and protein levels and disrupted localization of integrin β1 in claudin-7 KD cells. a Real-time RT-PCR was performed on integrins β1, β2, and β3. The mRNA level of integrin β1 was significantly reduced in claudin-7 KD cells. *P < 0.05. b Western blots show that integrin β1 and phospho-FAK levels were decreased in both claudin-7 KD cells (left) and claudin-7 knockout mouse lungs (right). At least six independent experiments were performed. c Immunofluorescence staining shows the localization of claudin-7 and integrin β1 in control and KD cells. Z-stack shows the co-localization of claudin-7 with integrin β1 at the basolateral surface in the merged image (top). In contrast, the localization of integrin β1 was greatly disrupted in the claudin-7 KD cells; it was no longer at the cell membrane (bottom). Arrows in control cells indicate the partial co-localization of claudin-7 with integrin β1. d Claudin-7 co-immunoprecipitated with integrin β1. Control cells were lysed in RIPA buffer without SDS and immunoprecipitated with either integrin β1 or claudin-7 antibody and then probed with either claudin-7 or integrin β1. e Control and KD cells were treated with anti-integrin β1 adhesion-blocking antibody or mouse IgG as a control. Cells were treated with the antibodies for two days, then cells were trypsinized and replated. The unattached cells were washed away two hours after replating, and the cells attached to the culture plate were trypsinized and counted. P values were shown above the bars on the right