Fig. 7. Frizzled colocalizes with vinculin and co-immunoprecipitates with vinculin from NP-40 detergent-insoluble cell extracts. Uninfected (A-C), Ad-ß-gal-infected (D-F) and Ad-Pax3^flag infected (G-H) paraformaldehyde (cystoskeletal)-fixed cells were dual labeled with anti-Frizzled (A,D,G) and anti-vinculin (B,E,H) antibodies. Confocal images of dual-stained cells shows near perfect overlap of Frizzled with vinculin at focal adhesions in control cells. In cells expressing Pax3 there was a reduction of Frizzled signals at many vinculin-containing focal adhesions (arrow in G and H); in these cells Frizzled signals overlapped extensively with vinculin in cytoplasmic vesicles (arrowhead in G-I). Strong nuclear staining of Frizzled and vinculin appeared to be an artifact of paraformaldehyde-cytoskeletal fixation. The subcellular distribution of Frizzled was examined by western blots of uninfected Saos-2 cell extracts fractionated into NP-40 soluble (S) and cytoskeletal-associated insoluble (I) extracts (J). (K) Frizzled co-immunoprecipitates with vinculin (lanes 7) and vice versa (lane 8) from insoluble extracts but not from soluble extracts (lanes 4 and 5, respectively) of uninfected Saos-2 cells. Dishevelled antibodies co-immunoprecipitate both vinculin and Frizzled from insoluble extracts (lane 9). Lane 1 represents control immunoprecipitation (IP) of insoluble extracts with IgG. Lane 2 represents 12.5% of input soluble extract and (lane 3) represents 25% of input insoluble extract used for IP. Bars, 20 µm (A,D,G).