Figure 2.

Agonist-dependent interaction between endogenous Gβγ and Rab11 and effect on their subcellular localization. Rab11 was immunoprecipitated from serum-starved HEK-293T cells grown to 80% confluence and stimulated with 10 μM LPA or left without stimulus (NS) as indicated. (A) The presence of Gβ, Rab11, and AKT in Rab11 immunoprecipitates and total lysates was detected by Western blot analysis (IP:Rab11 and total lysates, respectively). Control refers to immunoprecipitation performed with anti-Grb2 antibody instead of the anti-Rab11 antibody. (B) Left, confocal images showing the distribution of Gβ and Rab11a upon LPA stimulation. HEK-293T cells were serum starved and stimulated with 10 μM LPA for the indicated times or left unstimulated (NS), fixed and stained with anti-Gβ antibody and anti-Rab11 antibody as described in Materials and Methods and analyzed by confocal microscopy. Confocal images show the distribution of Gβ in NS cells or at selected times after stimulation with 10 μM LPA. Gβ distribution in NS cells is consistent with its presence preferentially on the cell surface and in some Rab11-positive vesicles. Stimulation with LPA for 5 or 15 min promoted Gβ internalization, which was detected in Rab11-positive endosomes. Bars, 8 and 10 μm, respectively. Right, percentage of colocalization area was determined using the public domain NIH ImageJ (http://rsb.info.nih.gov/nih-image/). NS cells, gray bar. LPA-stimulated cells, black bars.