Fig. 1. Exocyst expression, assembly and localization in non-metastatic and metastatic prostate tumor cells. (A) Dunning rat R3327-5'A (`A') and R3327-5'B (`B') cells were extracted in 1% Triton X-100. Extracts were subjected to immunoprecipitation with antibodies to Sec8. Presence of Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70 and Exo84 in equivalent amounts of whole cell extracts (`total') and precipitated immune complexes (`Sec8') was assessed by SDS-PAGE followed by immunoblotting with specific antibodies. (B) Subconfluent cultures of R3327-5'B, R3327-5'A or R3327-5'A cells stably expressing human E-cadherin were cultured on type I collagen-coated coverslips, and then processed for immunofluorescent staining with antibodies to Sec6 or Sec8, as described in the Materials and Methods. Samples were viewed with a Nikon Microphot-FX microscope (63x objective) and epifluorescent digital images were obtained using a Kodak DCS 760 digital camera. Arrows indicate accumulations of Exocyst proteins in protrusive extensions of R3327-5'A cells. (C) Sub-confluent cultures of R3327-5'A cells were cultured on Matrigel-coated coverslips, and then processed for immunofluorescent staining with phalloidin (to label f-actin) and antibodies to Sec6. Arrows indicate an accumulation of Exocyst within an actin-rich invadopodium. Scale bars: 20 µm.