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These findings unveil a new mechanism that enriches the diversity of MVB sorting pathways for signaling receptors. This work also suggests that ubiquitination of mammalian GPCRs is likely to have additional functions besides mediating receptor sorting at the MVB.

HRP-conjugated goat—anti rabbit and goat—anti mouse antibodies were purchased from Bio-Rad Laboratories. Alexa Fluor , , and secondary antibodies were purchased from Invitrogen.

Hanson Washington University, St. Louis, MO. Cells were grown and maintained as previously described Trejo et al. Human endothelial EA.

HeLa cells or endothelial cells were transfected with nM siRNA using either Lipofectamine Invitrogen or Oligofectamine Invitrogen , respectively, and examined after 48 h.

Cells were washed and preincubated on ice for 1 h with anti-FLAG antibody to label only receptors at the cell surface.

Equivalent amounts of lysates were used for analysis by immunoblotting. Endothelial cells were plated in 6-cm dishes 3. PAR1 degradation was assessed as previously described Russo et al.

After 48 h, cells were treated with agonist and lysed in buffer, as previously stated. Fluorescent images of 0.

The following protein sequences were acquired from the National Center for Biotechnology Information protein database: Rattus norvegicus available from GenBank under accession no.

Sequences were aligned using ClustalX 2. HeLa cells were plated in six-well culture dishes 4. Cells were placed on ice and incubated for 5 min with PBS, harvested, and gently permeabilized using 6.

Membranes were then divided into three aliquots either left untreated, treated with 2. The PCR amplification products were resolved by 1. HeLa cells were plated at 0.

An aliquot was removed, and the absorbance at nm was determined using a microplate reader SpectraMax Plus; Molecular Devices. Data were analyzed using Prism software version 4.

We thank members of the Trejo laboratory. We are grateful for reagents provided by Drs. Sundquist, P. Hanson, M. Martin-Serrano and acknowledge Drs.

Handel, J. Jones, D. Siderovski, and A. Marchese for reagents and advice. Author contributions: M. Dores and B.

Chen designed, performed, and analyzed the experiments and prepared the manuscript. Lin, U. Soh, M.

Paing, W. Montagne, and T. Meerloo contributed experimental work. Trejo contributed to project design and manuscript preparation.

Immunolabeled cells were processed and imaged by confocal microscopy. The colocalization of PAR1 and LAMP1 is shown in yellow in the merged image and is representative of many cells examined in three independent experiments.

Insets show magnifications of boxed areas. Cells were fixed and processed for immuno-EM. Dual-labeled ultrathin sections revealed that PAR1 WT and 0K mutant detected with nm gold-conjugated secondary antibody black arrows sorted to ILVs present in CDpositive MVBs, which was detected with 6-nm gold-labeled secondary antibody white arrows.

Bar, nm. The asterisk indicates a nonspecific band. Cell lysates were immunoblotted with anti-GFP and -actin antibodies.

The insets are magnifications of the boxed areas. Cell lysates were immunoblotted with anti-HA and -actin antibodies. Cell lysates were immunoblotted with anti-HA and -actin antibodies as controls.

Equivalent amounts of cell lysates were immunoprecipitated IP and examined as described in Fig. IB, immunoblotting.

Equivalent amounts of cell lysates were immunoprecipitated and analyzed as described in Fig. PAR degradation was assessed by immunoblotting IB.

Endogenous PAR1 was detected by immunoblotting. Cell lysates were immunoblotted with anti-ALIX and -actin antibodies.

The conserved residues of the YPX 3 L motif are shaded in gray. Cell lysates were immunoprecipitated and examined as described in Fig.

Pulldowns PD were analyzed for the presence of bound protein by immunoblotting. Input was analyzed by Coomassie staining. PAR1 degradation was analyzed as described in Fig.

Membranes were isolated and treated with proteinase K or proteinase K with 0. Sign In or Create an Account.

Advanced Search. User Tools. Sign In. Article Navigation. Article April 30 Dores , Michael R. This Site.

Google Scholar. Buxin Chen , Buxin Chen. Huilan Lin , Huilan Lin. Unice J. Soh , Unice J. May M. Paing , May M. Louis, MO William A.

Montagne , William A. Timo Meerloo , Timo Meerloo. Correspondence to JoAnn Trejo: joanntrejo ucsd. Author and Article Information.

Buxin Chen. Huilan Lin. Timo Meerloo. JoAnn Trejo. Received: October 06 Accepted: March 22 Online Issn: J Cell Biol 3 : —

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