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. 2010 Dec 20;31(5):1066–1075. doi: 10.1128/MCB.00883-10

FIG. 5.

FIG. 5.

Peptide disruption of the β-arrestin 1/ARHGAP21 complex in HEK AT1AR cells. (A) Sequences of the cell-permeant ARHGAP21/β-arrestin 1 disruptor peptide (peptide 842) based on the sequence of ARHGAP21 (amino acids 1331 to 1355) and a scrambled control peptide. (B) (Top) Immunoprecipitates (IP) of β-arrestin 1 from AT1AR HEK 293 cells pretreated with peptide (Pep.) 842 or a peptide control for the indicated times were blotted for ARHGAP21 before or after treatment with angiotensin II (AngII) (100 nM). (Center and bottom) Equal amounts of ARHGAP21 and β-arrestin 1 in inputs. (C) Densitometric analysis of the data shown in the four rightmost lanes of panel B. Results of 3 experiments are shown. **, P < 0.01. (D) (Top) RhoA activation in AT1AR HEK 293 cells as measured by a GST-Rhotekin pulldown assay following a time course of angiotensin II (100 nM) treatment with and without pretreatment (2 h) with peptide 842 or a control (con) peptide. +ve, positive; −ve, negative. (Bottom left) RhoA activation in AT1AR HEK 293 cells as measured by a GST-Rhotekin pulldown assay following PDGF (20 ng/ml) treatment with or without pretreatment (2 h) with peptide 842 or a control peptide. (Bottom right) Quantification by densitometry of the data shown on the left. The results of 3 experiments are shown. *, P < 0.05. (E) (Top and center) Western blot analyses of HEKB2 293 cell lysates following isoprenaline (10 μM) treatment with or without pretreatment (2 h) with peptide 842 or a control peptide. Samples were blotted for the β2-adrenergic receptor (B2Ar) and the phospho-β2-adrenergic receptor (Ser345/346). (Bottom) Immunoprecipitates of β-arrestin 1 from AT1AR HEK 293 cells pretreated with peptide 842 or a peptide control were blotted for PDE4D.