Kinetic association of Cdc42 with the exocyst complex during large particle uptake. (A and B) Sec8 associates with Cdc42 during large particle uptake. 293T cells were cotransfected with Myc-Cdc42wt, Sec8-HA, and Sec3-FLAG and were incubated with 4.5 (A)- or 1.5 (B)-µm beads. At the indicated times, cells were lysed, and Myc-Cdc42 was precipitated from the cleared lysates using anti-Myc antibody resin. Input and immunoprecipitate (IP) samples were analyzed by SDS-PAGE and Western blotting. (C) Quantification of A and B. Densitometry was performed on Western blots, and the quantity of Sec8-HA that precipitated with Myc-Cdc42 was normalized to input quantities and divided by starting amounts to derive the fold Sec8 immunoprecipitate. This assay was repeated multiple times, yielding similar results, and data are shown from one representative experiment.