Fig. 8. Reduction in cell adhesion by AIP1 proteins correlates with their presence in PYK-2 immunoprecipitates. HEK293 cells were transiently transfected with SETA, AIP1, AIP1-717-784, AIP1-784Stop, AIP1Y319F c-Cbl and lacZ as a control, as indicated. Two days after transfection cultures were harvested and underwent an ECIS cell attachment assay under confluent conditions on the electrode, over a period of 9 hours (see Materials and Methods for details). The values at each time point for the lacZ-transfected cultures from each experiment were averaged, taken as background and subtracted from the other data sets. Ten values around each hour were then taken, averaged and the standard deviation calculated. The values for each curve were set to zero at the zero time point and changes in resistance over time plotted. After an initial settling period of 3 hours the graphs reflect changes in resistance as cells attach and reach a steady state. (A) The resistance of SETA and AIP1 or c-Cbl-transfected cells moved in opposite directions over time. While SETA increased the resistance of the cell layer up to +1500 ohm, AIP1 and c-Cbl decreased it to about -1500 ohm. Accordingly, SETA mediates a pro-adhesive effect on HEK293 cells, while AIP1 and c-Cbl negatively affect cell adhesion. Cell density at the time of transfection had no major influence on these effects. (B) AIP1-717-784 and AIP1Y319F induced no major change in cell adhesion, while AIP1 and AIP1-784Stop induced a reduction similar to that observed in AIP1 (A). While SETA cotransfection did not alter the pattern of AIP1 and AIP1-784Stop cell adhesion, it caused an increase in adhesion when co-transfected with AIP1Y319F, which suggests that it's effect was dominant over this mutant, which had no effect on its own. Interestingly, cotransfection of SETA and AIP1-717-784 caused a reduction in adhesion. Error bars are smaller than plot symbols at some time points.