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. Author manuscript; available in PMC: 2006 Apr 6.
Published in final edited form as: J Biol Chem. 2004 Oct 7;279(51):53643–53652. doi: 10.1074/jbc.M401438200

Figure 2.

Figure 2

Interaction of GST-GRK2-(45–178) with Gαq point mutants activated by AlF4−, the Q209L or the R183C mutation. (A) HEK-293 cells were transfected with EE tagged versions of Gαq point mutants and Gβ and Gγ constructs. Cells were lysed and binding to GST-GRK2-(45–178) in the presence (+) or the absence (−) of AlF4− was determined as described in Experimental Procedures. The (+) and (−) lanes represent 40% of the Gαq or Gαq mutant pulled down from the 125 μl of lysate. In these experiments we detect little to no binding of GST-GRK2-(45–178) to Gαq or Gαq point mutants in the absence of AlF4−. Underneath the representative western blot the percent of each Gαq mutant pulled down by GST-GRK2-(45–178) in the presence of AlF4− is compared to the control, which is the percent of Gαq pulled down by GST-GRK2 (45–178) in the presence of AlF4−, and is represented graphically as the percent of control + S.D. (B) HEK-293 cells were transfected with EE tagged versions of Gαq-Q209L point mutants and Gβ and Gγ constructs. Cells were lysed and binding of the QL mutants to GST-GRK2-(45–178) was determined as described in Experimental Procedures. Results are plotted as described in A. (C) HEK-293 cells were transfected with EE tagged versions of Gαq-R183C point mutants and Gβ and Gγ constructs. Cells were lysed and binding of the RC mutants to GST-GRK2-(45–178) was determined as described in Experimental Procedures. The lanes labeled “P” represent 40% of the Gαq or Gαq mutant that was present in the pulldown from 200 μl of lysate. The lanes in A, B and C labeled “L” represent 4% of total Gαq or Gαq mutant available in the lysate for pull-down. Results are plotted as described in A. The (⋆) indicates that the amount of the marked Gαq mutant pulled-down is significantly different (p < 0.05) by one-way ANOVA followed by a Dunnett post-test, than the amount of Gαq pulled-down by GST-GRK2. The (⋆⋆) indicates that statistical analysis could not be performed on the binding of GST-GRK2-RH to the K77A-QL, P185K-QL or P185K-RC mutants because there was no detectable pull-down. The data are averages from three to six independent experiments.