Figure 2

(A) Dunning rat R3327-5′A prostatic tumor cells were seeded on 75 mm Transwell filters (3.0 μm pore size) and medium containing 10% FBS was added basolaterally to stimulate pseudopod extension, as described in Materials and Methods. An enriched pseudopod fraction was obtained by removing cell bodies from the top of filters with a cotton swab. Indicated proteins were identified by immunoblotting with specific antibodies, and protein levels were quantified using a Molecular Dynamics Typhoon phosphorimager. To determine the fold enrichment of each protein within pseudopods, values were normalized to protein levels present in an equivalent amount of whole cell extract. (B) Distribution of endogenous Sec6, Sec8, Sec15, Exo84, Syntaxin3, Syntaxin4, Munc18c and exogenous GFP in 5′A cells. Sub-confluent cultures of R3327-5′A cells were cultured on type I collagen, and then processed for immunofluorescent staining with indicated antibodies, as described in Materials and Methods. Bound antibodies were detected with appropriate FITC or Texas Red-conjugated secondary antibodies, and epifluorescence images were obtained. Arrows point to accumulations of membrane trafficking components at the tips of pseudopods. Note that accumulations of Sec15, Exo84 and GFP were never observed within pseudopods. Scale bar = 20 μm.