Figure 8. Recovery of cld7 and associated molecules in exosomes.

(A) Flow cytometry of latex beads bound wt, kd and rescue ASML exosomes stained with anti-cld7, -EpC, -Tspan8, -α6β4, -ezrin, -RhoA, -tubulin and -caveolin. Overlays of the negative control and stained samples are shown. (B) Lysates of ASML^wt, and -cld7^mPalm exosomes were precipitated with anti-cld7, -EpC, -CD44v6, -Tspan8 or -α6β4. Dissolved precipitates were separated by SDS-PAGE and were blotted with the indicated antibodies. The WB of exosome lysates is included as control. ASML^wt exosomes stain brightly with anti-Tspan8, -EpC, and -cld7 and distinctly with anti-α6β4, -ezrin, -RhoA and -tubulin. ASML-cld7^mPalm exosomes show reduced cld7⁺, -EpC⁺ and RhoA⁺, but more caveolin⁺ exosomes. Exosome lysate coimmunoprecipitation confirmed reduced recovery of EpC, CD44v6, α6β4 and Tspan8 in anti-cld7 precipitates of ASML-cld7^mPalm than ASML^wt exosomes.