Figure 2.
DAPLE binds preferentially to inactive versus active Gαs or Gαq. A, binding of DAPLE to the constitutively-active Gαs mutant R201C is diminished compared with Gαs WT. Lysates of HEK293T cells expressing Gαs WT, or Gαs R201C were incubated with GST or GST–DAPLE immobilized on GSH-agarose beads. Bead-bound proteins were detected by Ponceau S staining or IB as indicated. B, binding of DAPLE to Gαs loaded with GDP·AlF4− or with GTPγS is diminished compared with binding to Gαs loaded with GDP. Lysates of HEK293T cells expressing Gαs were incubated with nucleotides as indicated under “Experimental procedures” and incubated with GST or GST–DAPLE immobilized on GSH-agarose beads. Bead-bound proteins were detected by Ponceau S staining or IB as indicated. C, binding of DAPLE to the constitutively-active Gαq mutant Q209L is diminished compared with Gαq WT. Lysates of HEK293T cells expressing Gαq–HA WT or Gαq–HA Q209L were incubated with GST or GST–DAPLE immobilized on GSH-agarose beads. Bead-bound proteins were detected by Ponceau S staining or IB as indicated. D, binding of DAPLE to Gαq loaded with GDP·4− is diminished compared with binding to Gαq loaded with GDP. Lysates of HEK293T cells expressing Gαq were incubated with nucleotides as indicated under Experimental procedures” and incubated with GST or GST–DAPLE immobilized on GSH-agarose beads. Bead-bound proteins were detected by Ponceau S staining or IB as indicated. All results presented in this figure are representative of two independent experiments (n = 2).