Cancer du sein et micro-environnement tumoral: rôle de la protéase ...

Figure 3. Cath-D interacts with LRP1b in cellulo(A)Co-transfected cath-D and LRP1b co-immunoprecipitate. COS cells weretransiently co-transfected with LRP1b in the presence or the absence of cath-D orD231Ncath-D. Cath-D, LRP1b, and non-immune IgG (control)immunoprecipitations (IP) and cell extracts (CE) were analyzed by anti-cath-D(panel a) and anti-LRP1b (panel b) immunoblotting. Arrows show coimmunoprecipitatedcath-D.(B) Expression of LRP1 in fibroblasts and breast cancer cells. Protein expressionof LRP1b chain is shown in panel a. Whole cell extracts were subjected to SDS-PAGE and immunoblotting with the anti-LRP1b hybridoma. LRP1b transfectedCOS lysate was used as a positive control, LRP1-deficient MEF cells as anegative control, and b actin as a loading control.(C) Co-purification of endogenous LRP1 with cath-D. Panel a shows the copurificationof endogenous LRP1 with cath-D in cath-D-transfected MEFs. Elutedfractions were subjected to SDS-PAGE and immunoblotting with the anti-cath-Dantibody (top panels) and anti-LRP1b hybridoma (bottom panels). Panel b showsthe co-purification of endogenous LRP1 with cath-D in HMF cells treated withcath-D. HMF fibroblasts were incubated for 48h with conditioned mediumcontaining 15 nM of pro-cath-D. HMF cell extracts were purified on an M1G8-coupled column and analyzed by immunoblotting as described in panel a.