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32P incorporation was determined with a Storm Phosphorimager.
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50
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0344072503
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note
-
Transient transfection of HEK-293 cells was performed using a modified calcium phosphate precipitation method (31). COS-7 cells were transiently transfected with Lipofectamine (9). Monolayers of transfected cells were incubated in serum-free medium for 16 to 20 hours before stimulation (9).
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51
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0344072500
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-
note
-
2AR and Texas Red-labeled c-Src fluorescence were done with dual excitation (488 and 568 nm) and emission (515 to 540 nm, fluorescein; 590 to 610 nm, Texas Red) filter sets. Specificity of labeling and absence of signal crossover were established by examination of single-labeled samples.
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52
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0344072502
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note
-
Cells in 100-mm dishes were stimulated at 37°C in phosphate-buffered saline (PBS) containing 10 mM Hepes (pH 7.4), as described in the figure legends. Stimulations were terminated by the addition of dithiobis[succinimidylpropionate] (DSP) to a final concentration of 2 mM, and plates were rocked for 30 min at room temperature. Cells were washed three times by centrifugation at 4°C with PBS/Hepes to remove unreacted DSP, then lysed in RIPA buffer (10) before immunoprecipitation.
-
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53
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0345365944
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note
-
6-tagged β-arrestin 1 and c-Src resolved along with the receptor immunoprecipitates.
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54
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0344934537
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note
-
Flag epitope-tagged truncation or deletion mutants of β-arrestin 1 were prepared by the polymerase chain reaction (PCR), incorporating an Eco RI site, minimal Kozak sequence (ACC), and initiator methionine codon into the 5′ primer, and the Flag epitope sequence, stop codon, and an Xho I site into the 3′ primer. PCR products were subcloned into a peptide minigene expression cassette as described (33). DNA sequences were confirmed by dideoxynucleotide sequencing.
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55
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0344072501
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note
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6-tagged β-arrestin 1 and coprecipitated Src-GST fusion proteins were detected simultaneously by protein immunoblotting with rabbit polyclonal anti-GST-β-arrestin 1. Competition for binding between c-Src and GST-Src SH2 or GST-Src SH3 was performed with the GST-Src fusion protein in 20-fold excess.
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56
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0344072499
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note
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125]pindolol.
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57
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0345365942
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note
-
Aliquots of whole-cell lysate from appropriately stimulated cells (30 μg of protein per lane) were resolved by SOS-PAGE, and Erk 1 and Erk2 phosphorylation was detected by protein immunoblotting with rabbit polyclonal phospho-MAP kinase-specific IgG. Quantitation of Erk 1 and Erk2 phosphorylation was performed with a Storm Phosphorimager. After quantitation of Erk 1 and Erk2 phosphorylation, nitrocellulose membranes were stripped of Ig and reprobed with rabbit polyclonal anti-Erk 2 IgG to confirm equal loading of Erk protein.
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0344934535
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note
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R.J.L. and M.G.C. are investigators with the Howard Hughes Medical Institute. Supported in part by NIH grants DK02352 and DK55524 (L.M.L.), HL 16037 (R.J.L.), NS19576 (M.G.C.), and Heart and Stroke Foundation of Ontario Grant NA3349 (S.S.G.F.). G.J.D.R. is supported by NIH Medical Scientist Training Program Grant T32GM-07171. We thank M. J. Eck for purified recombinant c-Src and D. Addison and M. Holben for excellent secretarial assistance.
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