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. 2012 Dec 7;7(12):e50557. doi: 10.1371/journal.pone.0050557

Figure 5. Biology of alpha arrestins.

Figure 5

(A) Ubiquitination effect of alpha arrestins on GPCR. Empty vector, pBSR-ARRDC3-Flag, or pBSR-ARRDC3-Flag PY motif mutant construct was cotransfected with pcDNA3-HA-b2AR-V5. After 24 h incubation, cells were serum-starved for 2 h and treated or not with 1 uM Iso for 30 m. The transfected cells were lysed, and lysates were immunoprecipitated (IP) and analyzed by western blot (WB). The same experiment was performed with ARRDC4 and V2R (1 uM AVP treatment for 30 m). (B) The mean values (+/− S.D.) of three independent experiments are included in the histogram (p value levels and lines used as in Fig. 2). (C.) Effects of siRNA-mediated ADDRC3 knockdown on b2AR ubiquitination and ITCH expression. Scrambled siRNA, and ARRDC3 siRNAs #1 and #2 were cotransfected with HA-b2AR-V5 construct. After 48 h incubation, cells were serum-starved for 2 h and treated with 1 uM Isoproterenol Iso for 30 m. The transfected cells were lysed, and the lysates were immunoprecipitated (IP) and analyzed by western blot (WB). (D) Histograms of mean (+/− S.D.) and p-values calculated from three different experiments by paired, two-tailed t-tests comparing ligand-stimulated cells transfected with ARRDC3 siRNA versus with control siRNA (p value levels used as in Fig. 2).