Figure 4.
Analysis of paxillin content of SAM by Western blotting. (A) Serum-starved Swiss 3T3 cells were permeabilized with SL-O for 8 min in the presence of 1 mM GDP at 37°C. After this, 1 mM Mg ATP was added in the absence (0) or presence of 50 μM GTPγS or 50 μg/ml GTP-loaded Val14rhoA for 8 min. In some cases, 0.2 μg/ml C3-transferase was included with 0.5 mM NAD+ during the permeabilization period, and before addition of GTPγS. The paxillin content of SAM from duplicate coverslips was then determined by Western blotting. (B) Cells were permeabilized for various times and then challenged with 1 mM Mg ATP in the absence or presence of GTPγS (as in Fig. 2, C and D). SAM was then prepared by wet cleavage at 4°C. The vinculin (○) and paxillin (•) contents of SAM were determined by Western blotting and a quantitative estimate of these obtained by densitometric scanning of the blots. The basal values (ATP alone) were then subtracted from those found in the presence of GTPγS (ATP + GTPγS) and plotted as GTPγS-stimulated recruitment. These data represent the mean (± SEM) of values obtained from three separate experiments. (C) Quantitative analysis of the paxillin content of SAM from experiments such as that displayed in A. These data represent the mean (± SEM) of values obtained from six separate experiments.