Figure 4. Intramembrane interactions of CD82 with β₁ integrins are essential for CD82 inhibition of fibronectin-induced EMT.

A. PZ-HPV-7 prostate epithelial cells were lysed with Brij 97 detergent, and immunoprecipitation (IP) was performed with normal mouse IgG or anti-CD82 antibody. The immunoprecipitates were analyzed by immnublotting using anti-integrin β₁, α₃, α₅, or α₆ antibody. B. CD82 mutant cDNA, which encodes CD82 with a large extracellular loop (LEL) substituted with that of TM4SF2 as illustrated, was generated by PCR and subcloned into the pAdEasy-1 adenoviral vector to produce recombinant adenovirus. C. CD82-deficient PC3 prostate cancer cells grown on fibronectin (FN) were infected with adenovirus containing a wild-type (wt) or mutant (mt) CD82 expression construct, and Brij 97 detergent lysates were subjected to immunoprecipitation with an anti-β₁ integrin antibody followed by immunoblotting analysis using antibodies that recognize the C-terminus or LEL of CD82 and the LEL of TM4SF2. D. PC3 cells grown on poly-L(+)-lysine (p-Lys) or FN were infected with adenovirus containing a wt- or mt-CD82 expression construct and then assessed for the protein levels of E-cadherin and Snail. E. PC3 cells grown on FN were infected with wt-CD82 construct-containing adenovirus either alone or together with mt-CD82 construct-containing adenovirus and examined for E-cadherin and Snail expression. Numbers in parentheses represent the MOI values of adenovirus.