Figure 8.

Turnover of phosphoproteins at focal complexes is reduced in the absence of RACK1. (A) GFP-dSH2 was co-transfected with pooled control (Csi) or RACK1 (Rsi) siRNA in HEK cells. Cells were plated on 3 μg/ml fibronectin and imaged by time-lapse fluorescence microscopy. Representative frames are shown from time-lapse sequences used to quantify duration of adhesions. For each sequence, T= 0 min is the first frame in which the adhesion marked by the arrow was observed. Bar, 3 μm. (B) Quantification of the persistence of GFP-dSH2 in focal complexes demonstrates that phosphoproteins remain at focal complexes for longer durations in the absence of RACK1. Duration measurements were made by counting the amount of time elapsed between the first and last frames in which an individual adhesion was observed in HEK cells that express control siRNA (Csi) or RACK1 siRNA (Rsi). The graph represents the average from at least three separate experiments, with error bars represented as the mean ± SEM. (C) HEK cells were transiently transfected with dSH2-GFP and either control (Csi) or RACK1 (Rsi) siRNA for 48 h. Immunoprecipitation for GFP or nonspecific IgG control was performed as described in Materials and Methods and immunoblotted for paxillin. Total cell lysates from control and RACK1-deficient cells were immunoblotted for phosphopaxillin (pY118), total paxillin and RACK1 as indicated. Representative blots from three separate experiments are shown.