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Fig. 3. Exocyst colocalizes with focal complexes at pseudopod tips of migrating prostate tumor cells. (A) Distribution of endogenous Sec6 and paxillin in R3327-5'A prostatic tumor cells. Cells were seeded on fibronectin-coated glass coverslips for 18 hours, then were fixed with 2% paraformaldehyde, permeabilized with 1% Triton X-100, incubated with mouse anti-Sec6 (mAb 9H5) and rabbit anti-paxillin antibodies, then stained with FITC-conjugated goat anti-mouse and Texas Red-conjugated donkey anti-rabbit IgG. Epifluorescence images were obtained as described in Fig. 1. Arrows indicate tips of protrusive pseudopods, within which Sec6 and paxillin appear to colocalize. In lower panels, arrowheads indicate structures within pseudopods that stained with anti-paxillin antibodies, but not anti-Sec6 antibodies, and asterisks indicate structures that stained with anti-Sec6 antibodies but not anti-paxillin antibodies. (B) Distribution of endogenous Sec6, Git1, Nck1/2 and β-PIX in 5'A cells. Cells were cultured, fixed and permeabilized as described in the Materials and Methods. Sec6 distribution was compared with that of Git1, β-PIX and Nck1/2. Sec6/Git1 and Sec6/β-PIX images were collected by epifluorescence microscopy. Sec6/Nck1/2 images were obtained with a Zeiss confocal laser-scanning microscope (63x objective) using a krypton/argon laser with 488 nm (FITC) and 568 nm (Texas Red) laser lines. (C) Specificity of antibodies. 5'A cells were lysed and 
1 µg of protein was loaded per lane onto a 10% SDS-PAGE gel. The proteins were transferred to Immobilon PVDF membranes and incubated with rabbit polyclonal antibodies to paxillin, GIT1 or Nck1/2 or mouse mAb to β-PIX. Blots were then probed with HRP-conjugated secondary antibodies and developed for ECL detection.
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