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. Author manuscript; available in PMC: 2013 Apr 1.
Published in final edited form as: Nat Cell Biol. 2012 Sep 2;14(10):1079–1088. doi: 10.1038/ncb2568

Figure 2. AGB1 is essential for AtRGS1 internalization.

Figure 2

(A) AtRGS1-YFP was transiently expressed in an informative set of G-protein mutants and treated with 6% glucose. Internalization of AtRGS1-YFP was imaged in Col-0, rgs1-2, gpa1-3, agb1-2, gpa1-4 agb1-2 double null mutants (αβ-null) and gpa1-4 agb1-2 agg1-2 agg2-1 quadruple null mutants (αβγ-null) without and with 6% glucose. (B) Quantitation of percent AtRGS1-YFP fluorescence (FL) internalized in epidermal cells transiently expressed before and after glucose stimulation. Error bars = SEM, n = 5. (C) Seedlings of Col-0, gpa1-3 and agb1-2 were treated with 200 mM cycloheximide (CHX). Relative steady-state levels of AtGPA1 and AGB1 protein in the seedling were analyzed by immunoblot analysis with anti-AtGPA1 and anti-AGB1 antisera. (D) AtRGS1-YFP was transiently expressed in rgs1-2 null mutants, 35S::AtGPA1, 35S::AtGPA1(Q222L) (active mutant) or 35S::AGB1 lines and treated with 6% glucose. “35S::” represents a constitutive promoter from the Cauliflower Mosaic virus used for ectopic overexpression. (E) AtRGS1-YFP and 35S::AtGPA1 were transiently expressed in a gpa1-4 agb1-2 double mutant and treated with 6% glucose. (F) Both AtRGS1-YFP and 35S::AtGPA1(Q222L) were transiently expressed in the gpa1-4 agb1-2 double mutant and treated with 6% glucose (left panels). AtRGS1-YFP and 35S::AtGPA1(Q222L) were also transiently expressed in the gpa1-3 mutant (right panel). (G) AtRGS1(E320K)-YFP was transiently expressed in rgs1-2 null mutants, 35S::AtGPA1, 35S::AtGPA1(Q222L) (active mutant) or 35S::AGB1 lines then treated with 6% glucose prior to imaging.(H) Quantitation of percent AtRGS1-YFP and AtRGS1-E320K-YFP fluorescence internalized in epidermal cells before and after 6% glucose stimulation. Error bars = SEM, n = 5. The genetic background is indicated: rgs1-2, ectopic expression of AtGPA1 (35S-GPA1 in gpa1-4 null background), ectopic expression of constitutively-active AtGPA1 (35S::GPA1(Q222L) in gpa1-4 null background), and ectopic expression of AGB1 (35S::AGB1 in agb1-2 null background). (I) Quantitation of percent of AtRGS1-YFP fluorescence in epidermal cells transiently expressing the AtGPA1 in the gpa1/agb1 double mutant (35S::AtGPA1 in gpa1-4 agb1-2 null background), constitutively-active AtGPA1 (35S::GPA1-QL) in the gpa1/agb1 double mutant), and 35S::GPA1-QL in the gpa1-3 mutant). Error = SEM, n = 5.All scale bars = 10 µm. GPA1-QL represents GPA1(Q222L). Quantitation of fluorescence is described in Methods.