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Subcellular colocalization and coIP of alpha arrestins with activated GPCR, clathrin, and endosome markers.

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posted on 07.12.2012, 02:28 by Fortune F. Shea, Jennie L. Rowell, Yechaowei Li, Tien-Hsien Chang, Carlos E. Alvarez

(A) Subcellular colocalization of alpha-arrestin-mCherry in receptor HA-b2AR permanent cell lines. HEK-293T cells stably expressing HA-b2AR were transiently cotransfected with ARRDC3-mCherry construct. 24 h after transfection, cells were serum-starved for 2 h, treated or not with 1 uM isoproterenol (Iso) ligand for 30 m, and then washed, and fixed and permeabilized. HA-b2AR was stained with rabbit anti-HA antibodies followed by incubation with Alexa Fluor 488-conjugated goat anti-rabbit secondary antibody. Epifluorescence images were captured using AxioVision or Zeiss 510 META confocal microscope (receptor, green; aArr, red). (B) Co-immunoprecipitation of aArr ARRDC3 with b2AR, clathrin and Hrs. HEK-293T cells were transiently cotransfected with HA-b2AR-V5 plus either empty vector, pBSR-ARRDC3-GFP, or pBSR-ARRDC3 PY motif mutant construct respectively. After 24 h incubation, cells were serum-starved for 2 h and treated or not with 1 uM Iso for 30 m. The cells were lysed, and lysates were immunoprecipitated (IP) and analyzed by western blot (WB). (C) Co-immunoprecipitation of aArr ARRDC4 with V2R, clathrin, and Hrs. HEK-293T cells were transiently cotransfected with HA-V2R-V5 plus either empty vector, pcDNA3-ARRDC4-Flag, or pcDNA3-ARRDC4 PY motif mutant construct respectively. After 24 h incubation, cells were serum-starved for 2 h and treated or not with 1 uM AVP for 30 m. The cells were lysed, the lysates were immunoprecipitated (IP), and analyzed as above. (D, E) Histograms of B and C from three independent experiments including the mean (+/− S.D.) and p-values calculated by paired, two-tailed t-tests on signal from untreated and ligand-treated samples versus their respective vector control are denoted by thin lines and treated vs. untreated samples are denoted by bold lines (***, p<0.001; **, p<0.01; *p<0.05). (F) Subcellular colocalization of alpha-arrestin-mCherry with early (Rab5) and late (Rab7) endosomal markers in HA-V2R-V5 permanent cells. HA-V2R-V5 permanent cells were transiently co-transfected with ARRDC3- or ARRDC4-mCherry construct with Rab5- or Rab7-GFP construct. After 24 h, transfected cells were serum-starved, treated or not with 1 uM AVP for 30 m, and fixed with 4% paraformaldehyde. Epifluorescence images were captured using AxioVision confocal microscope (aArr, red; Rab, green).

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