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Figure 5: Assignment of signaling molecules associated with CD44 and CD44v6 in cells and TEX. (a) List of signal transduction-engaged proteins in cells coimmunoprecipitating with CD44 (red) or CD44v6 (green) (Reactome analysis); STRING pathway analysis of CD44- and CD44v6-associated proteins in (b) TEX and (c) cells and TEX (CD44 framed violet, integrins: framed green, tetraspanins: framed red; full names of synonyms: Table S6). (d-f) Tspan8-dependence of CD44v6-associated molecules on recovery in TEM and transfer into TEX. (d) Flow cytometry analysis of CD44v6- and/or Tspan8-associated molecules in cells and TEX; mean ± SD of 3 experiments; significant differences between wt and CD44v6kd cells/TEX: , significant differences between wt and Tspan8kd cells/TEX: s. (e) WB of wt, CD44v6kd, and Tspan8kd cell lysates after sucrose gradient fractionation and blotting with the indicated antibodies; a representative example and relative band intensity ± SD of 3 experiments evaluated by ImageJ are shown; significant differences between wt and CD44v6kd lysates: , significant differences between wt and Tspan8kd lysates: s. (f) WB of wt, CD44v6kd, and Tspan8kd cell and TEX lysates with anti-CD44 and anti-Tspan8 after coimmunoprecipitation with the indicated antibodies. A representative example of 3 assays is shown. A significantly higher number of signaling-engaged proteins coimunoprecipitated with CD44v6 than CD44 in cells and TEX. Dominant in CD44v6-precipitates are components of integrin, EPH-Ephrin, cytokine, GPCR, and NOTCH signaling pathways. The majority of proteins, particularly those recovered in TEX, also associate with Tspan8. This suggests that the association with CD44v6 partly relies on Tspan8-mediated TEM recruitment.