Fig. 2

TSPAN3 interacts with—and upregulates—β1 integrin. A Ectopically expressed tetraspanin 3 (TSPAN3/T3) interacts with β1 integrin in A549 cells. 48 h post transfection of pCMV6-Myc-FLAG-TSPAN3 plasmid, cell lysates were immunoprecipitated with anti-FLAG antibodies or control IgG and then subjected to western blotting with anti-β1 integrin and anti-TSPAN3 antibodies. B Coimmunoprecipitation revealed that endogenous TSPAN3 interacts with β1 integrin in A549 cells. Cell lysates were immunoprecipitated with anti-TSPAN3 antibody or control IgG and then subjected to western blotting using anti-β1 integrin (ITGβ1) and anti-TSPAN3 antibodies. C Schematic of TSPAN3 LEL domain deletion. D Immunoprecipitation study indicates that TSPAN3 interacts with β1 integrin via the LEL domain. A549 cells were transfected with wild-type (WT) GFP-TSPAN3 or TSPAN3-ΔLEL mutant plasmids. After 48 h of transfection, cell lysates were immunoprecipitated with the anti-GFP antibody; the presence of β1 integrin was measured by western blotting with the anti-β1 integrin antibody. E Western blotting shows that TSPAN3 knockdown downregulated the expression of β1 integrin in A549 and H460 cells. Overexpression of TSPAN3 had the opposite effect. GAPDH served as the loading control (n = 3 independent experiments). F Overexpression of wild type TSPAN3 resulted in upregulated expression of β1 integrin, whereas overexpression of the TSPAN3-ΔLEL mutant did not exhibit any such effect. GAPDH served as the loading control (n = 3 independent experiments).G The association between TSPAN3 and β1 integrin investigated using immunohistochemistry (magnification ×200). IB, immunoblotting; IP, immunoprecipitation