Colocalization of PC-PLC and HER2 on plasma membrane of SKBr3 cells. (a) CLSM detection of PC-PLC and HER2 in either unfixed (top and middle panels) or fixed and permeabilized SKBr3 cells (bottom panel) by using rabbit polyclonal α-PC-PLC (green) and α-HER2 W6/100 mAb (red). Colocalization areas are represented in yellow. The middle panel shows the tridimensional reconstruction of PC-PLC and HER2 expression on the plasma membrane. Scale bars, 8 μm. At least five independent series of experiments were performed for each condition. (b) PC-PLC immunoblotting analyses of immunoprecipitated biotinylated proteins isolated from SKBr3 cells, detected by streptavidin-HRP (left, IB:STREP HRP) or by α-PC-PLC (right, IB:α-PC-PLC). TL, total lysate; bTL, biotinylated total lysate; CTR IgG, control for α-PC-PLC IgG; IP α-PC-PLC, α-PC-PLC immunoprecipitates. *IgG heavy chains. (c) Sucrose gradient fractions isolated from SKBr3 cell lysates and analyzed by Western blotting for HER2 (185 kDa) and PC-PLC (66 kDa) detection. T, total cell lysate. (d, e) Western blot analyses of α-HER2 and α-PC-PLC (d) and α-EGFR and α-PC-PLC (e) immunoprecipitates (IP), blotted with the mutual Abs (α-PC-PLC, α-HER2, and α-EGFR, respectively), compared with the respective controls (CTR IgG α-PC-PLC, CTR IgG α-HER2, CTR IgG α-EGFR). Panels b, c, d, show representative results of three independent experiments. The immunoprecipitation in panel e was repeated twice.