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Fig. 1. Knockdown of cortactin expression attenuates transferrin uptake. (A) Cells grown in a six-well plate were transfected with cortactin siRNAs H1, H2, H3 and GFP siRNA as described in Materials and Methods. After 24 hours of transfection, cortactin expression in the treated cells was examined by immunoblot analysis. The same samples were also analyzed for actin and dynamin expression as negative controls. (B) Cells transfected with cortactin siRNA H1 (a and b), H2 (c and d), H3 (e and f) and GFP siRNA (g and h) were incubated with Oregon Green-labeled transferrin at 37°C for 30 minutes, fixed and stained with cortactin antibody (a,c,e and g) and DAPI (b,d,f and h). Internalized transferrin was seen as green dots in the cytoplasm. Magnification x600. (C) Percentage uptake of transferrin was quantified based on the fluorescence intensity of internalized transferrin in treated cells. The data shown are mean ± s.e.m. (n>400).
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