1. Identification of β-Arrestin2 as a G Protein-Coupled Receptor-Stimulated Regulator of NF-κB Pathways 
Hua Gao, Yue Sun, Yalan Wu, Bing Luan, Yaya Wang, Bin Qu, Gang Pei 
Molecular Cell 
Volume 14, Issue 3, Pages (May 2004)
DOI: /S (04)

2. Figure 1 Identification of IκBα as β-Arrestin2-Interacting Molecule
(A) Interaction of β-arrestin2 and IκBα in vitro. Purified GST, GST-β-arrestin2, and His-IκBα were combined in vitro for His or GST pull-down assay as indicated. Immunoblots depict GST-β-arrestin2 coimmunoprecipitated with His-IκBα
(B and C) Binding of β-arrestin2 to IκBα in mammalian cells. β-arrestin2 or β-arrestin1 and IκBα were expressed together in cells. Proteins immunoprecipitated with Flag or HA-specific antibody or cell lysate were subject to immunoblotting with antibody against HA or Flag as indicated.
(D) Association of endogenous β-arrestins and IκBα. IκBα or β-arrestins were immunoprecipitated with IκBα-specific antibody or anti-β-arrestins egg yolk IgY, and the precipitates were subjected to immunoblotting with antibodies against β-arrestins or IκBα. Immunoblot analysis of β-arrestins and IκBα in whole cell lysate was shown.
The data are representative of at least three similar experiments. βarr2, β-arrestin2; βarr1, β-arrestin1; IP, immunoprecipitated; IB, immunoblot.
Molecular Cell  , DOI: ( /S (04) )

3. Figure 2 Modulation of NF-κB Activation by β-Arrestin2
(A) Modulation of NF-κB DNA binding activity. Transfected with β-arrestin2 (left panel) and β-arrestin2 siRNA (right panel), respectively, HEK293 cells were induced by TNF-α (10 ng/ml) for the indicated times after 12 hr serum starvation, and then NF-κB and NF-1 DNA binding activities were examined by EMSA.
(B) Modulation of TNF-α-induced NF-κB transcription activity. HEK293 cells were cotransfected with pNF-κB-TA-Luc and increasing concentrations of β-arrestin2 (left panel) or β-arrestin2 siRNA (right panel), and then the cells were induced by TNF-α for an additional 6 hr before luciferase activity was determined.
(C) Modulation of NF-κB p65-induced NF-κB transcription activity. HEK293 cells were cotransfected with NF-κB p65 combined with pNF-κB-TA-Luc and β-arrestin2 (left panel) or β-arrestin2 siRNA (right panel), and luciferase activity was determined 36 hr later.
Data are means ± SE of four independent experiments. *p < 0.05, and **p < 0.01 versus control.
(D) Binding of β-arrestin2 to IκBα-NF-κB p65 complex. HEK293 cells were cotransfected with the indicated plasmids. Proteins immunoprecipitated with Flag-specific antibody were subject to immunoblotting with antibody against HA or Flag as indicated. The data are representative of four similar experiments.
Molecular Cell  , DOI: ( /S (04) )

4. Figure 3 Mapping the Interaction Regions between β-Arrestin2 and IκBα
(A) Schematic representation of wild type, N-terminal, and C-terminal truncation mutants of β-arrestin2.
(B) Mapping the IκBα binding region of β-arrestin2 using truncation mutants. Flag-IκBα was expressed with deletion mutants of HA-β-arrestin2 in HEK293 cells. Proteins immunoprecipitated with HA-specific antibody or cell lysate were subject to immunoblotting with antibody against Flag or HA as indicated.
(C) Schematic representation of wild type, N-terminal, and C-terminal truncation mutants of IκBα.
(D) Mapping the β-arrestin2 binding region of IκBα using truncation mutants. Flag-β-arrestin2 was expressed with deletion mutants of HA-IκBα in HEK293 cells. Proteins immunoprecipitated with Flag-specific antibody or cell lysate were subject to immunoblotting with antibody against HA or Flag as indicated.
The data are representative of four similar experiments.
Molecular Cell  , DOI: ( /S (04) )

5. Figure 4 β-Arrestin2 Stabilizes IκBα through Its Binding to IκBα
(A) Stabilization of endogenous IκBα in HEK293 cells induced by TNF-α. Cells were transfected with β-arrestin2, β-arrestin2(60-409), and β-arrestin2 siRNA, respectively, incubated for 48 hr or 96 hr, and then induced by TNF-α for the indicated times. Immunoblot analysis was done on the cell extracts to detect the phosphorylation and degradation of IκBα.
(B) IKK activity was assayed in an in vitro kinase assay. Immunoprecipitated Flag-IKKβ was combined with recombinant GST-IκBα(1-54), full-length IκBα, β-arrestin2, or GST in vitro for kinase assay as indicated. Flag-IKKβ protein amounts were determined by immunoblotting.
(C) Immunoblot analysis of NF-κB p65 translocation using nuclear extracts of HEK293 cells. Cells were transfected with β-arrestin2 and β-arrestin2 siRNA, respectively, incubated for 48 hr or 96 hr, induced by TNF-α for the indicated times, and then the nuclear extracts were separated to detect NF-κB p65 translocation.
(D) No effects of β-arrestin2(60-409) on NF-κB DNA binding activity. HEK293 cells were transfected with β-arrestin2 or β-arrestin2(60-409). After serum starvation for 12 hr, the cells were induced by TNF-α for the indicated times, and then NF-κB and NF-1 DNA binding activities were examined by EMSA.
Data are means ± SE of at least three independent experiments. *p < 0.05, and **p < 0.01 versus control.
Molecular Cell  , DOI: ( /S (04) )

6. Figure 5
Enhancement of β-Arrestin2-IκBα Interaction by Stimulation of β2AR
(A) Interaction of β-arrestin2 and IκBα is enhanced by stimulation of β2AR. HEK293 cells were cotransfected with β2AR, HA-β-arrestin2, and Flag-IκBα. After serum starvation for 12 hr, the cells were stimulated with Iso (10 μM) for the indicated times. Proteins immunoprecipitated with HA-specific antibody or cell lysate were subject to immunoblotting with antibodies against Flag or HA as indicated.
(B) Association of endogenous β-arrestin2 and IκBα is increased by Iso treatment. HEK293 cells were transfected with β2AR. After serum starvation for 12 hr, the cells were stimulated with Iso for the indicated times. Proteins immunoprecipitated with IκBα-specific antibody or cell lysate were subject to immunoblotting with antibodies against β-arrestins (A1CT) or IκBα as indicated.
(C) Pretreatment with β2AR-antagonist propranolol totally blocks the effect of Iso treatment. HEK293 cells were transfected with β2AR. After serum starvation for 12 hr, the cells were pretreated with propranolol (10 μM) and then stimulated with Iso. The coimmunoprecipitation was performed as in (B).
Results represent the mean ± SE of four independent experiments. *p < 0.05, and **p < 0.01 versus the basal.
(D) Colocalization of HA-β-arrestin2 and GFP-IκBα or Flag-β2AR and GFP-IκBα in HEK293 cells after Iso treatment for 1 min. HA-β-arrestin2 (red) and Flag-β2AR (red) were detected with HA or Flag antibody. Scale bar, 10 μm.
Molecular Cell  , DOI: ( /S (04) )

7. Figure 6
Stimulation of β2AR Also Improves the β-Arrestin2's Inhibitory Effects on NF-κB Activation
(A) Activation of β2AR stabilized endogenous IκBα in HeLa cells induced by TNF-α. Cells were transfected with β2AR. After serum starvation for 12 hr, the cells were induced by TNF-α combined with or without Iso for the indicated times. Immunoblot analysis was done on the cell extracts to detect the phosphorylation and degradation of IκBα.
(B) Immunoblotting analysis of NF-κB p65 translocation using nuclear extracts of HeLa cells. Cells were transfected with β2AR. After serum starvation for 12 hr, the cells were induced by TNF-α combined with or without Iso for the indicated times, and then the nuclear extracts were separated to detect the NF-κB p65 translocation.
(C) β-arrestin2 is required for β2AR signaling regulation of NF-κB pathways. β2AR was transfected into wild-type or β-arrestin-null MEF cells with or without β-arrestin2. After serum starvation for 12 hr, the cells were induced by TNF-α combined with or without Iso for the indicated times, and then NF-κB and NF-1 DNA binding activities were examined by EMSA.
Data are means ± SE of at least three independent experiments. *p < 0.05, and **p < 0.01 versus TNF-α alone.
Molecular Cell  , DOI: ( /S (04) )

8. Figure 7
β-Arrestin2 Regulates NF-κB Target IL-6, IL-8, and IL-1β Expression
(A) HeLa cells were transfected with β-arrestin2 siRNA, incubated for 96 hr, induced by presence or absence of TNF-α, and then the cells were subjected to ELISA and RT-PCR for detection of protein level and mRNA level of IL-6 and IL-8.
(B) THP-1 cells were transfected with β-arrestin2 siRNA, incubated for 96 hr, induced by presence or absence of LPS, and then the cells were subjected to ELISA and RT-PCR for detection of protein level and mRNA level of IL-6 and IL-1β.
(C) MEF cells were transfected with β-arrestin2 or β-arrestin2(60-409), incubated for 48 hr, induced by presence or absence of TNF-α, and then the cells were subjected to ELISA and RT-PCR for detection of protein level and mRNA level of IL-6.
(D) Model illustration of β2AR-stimulated β-arrestin2-IκBα interaction on NF-κB activation.
Results represent the mean ± SE of three independent experiments. *p < 0.05 versus control.
Molecular Cell  , DOI: ( /S (04) )