Fig. 6

Colocalization and coimmunoprecipitation of proteases with tetraspanins in wt- and ko-MCA cells. Confocal microscopy of MMP2, MMP9, MMP14 and TACE with (a) CD9 and (b) Tspan8 and CD151 in wt- and ko-MCA cells; representative examples of overlays with the indicated proteases and tetraspanins are shown; c precipitates of wt- and ko-MCA cell and TEX lysates with anti-MMP2, -MMP9, -uPAR and -TACE were separated by SDS-PAGE, blotting membranes with anti-CD9, -CD151 and -Tspan8; representative examples are shown; d lysates of wt- and ko-MCA tumors were fractionated by sucrose gradient centrifugation. After SDS-PAGE of fractions 1–4 and pooled fractions 5–8 and 9–12, membranes were blotted with anti-MMP2, -MMP9, -uPAR and -TACE; representative examples are shown. MMP2, MMP9 and MMP14 preferentially colocalize with Tspan8, TACE colocalizes with Tspan8 and CD151. However, in the absence of Tspan8 and CD151, proteases also associate with CD9, which was confirmed by coIP. Density fractionation indicated impaired invasion being a sequel of the missing association of MMP2 and MMP9 with Tspan8 and/or CD151 in TEM. Recovery of uPAR and TACE in light density fractions being not affected in Tspan8ko- and/or CD151ko-MCA cells points towards TEM-independent associations