alix-bro1  (Addgene inc)

Journal: The Journal of Cell Biology

Article Title: ALIX binds a YPX 3 L motif of the GPCR PAR1 and mediates ubiquitin-independent ESCRT-III/MVB sorting

doi: 10.1083/jcb.201110031

Figure Lengend Snippet: A YPX 3 L motif of PAR1 mediates binding to ALIX. (A) Alignment of PAR1 and PAR2 ICL2 sequences from various species. The conserved residues of the YPX 3 L motif are shaded in gray. (B) HeLa cells coexpressing HA-ALIX WT and either FLAG-PAR1 WT or Y206A mutant were stimulated with 100 µM SFLLRN at 37°C, as indicated. Cell lysates were immunoprecipitated and examined as described in . IB, immunoblotting. The data (mean ± SD) represent the amount of immunoprecipitated ALIX normalized to the amount of immunoprecipitated PAR1 and were significant, as determined by Student’s t test (**, P < 0.01; n = 3). A.U., arbitrary unit. (C) HeLa cells transfected with FLAG-PAR1 and either HA-ALIX WT or F676D mutant were stimulated with agonist and processed as previously described. Cell lysates were immunoblotted with anti-HA and -actin antibodies. The data (mean ± SD) were calculated, as previously described, and significant, as determined by Student’s t test (**, P < 0.01; n = 3). (D) Biotinylated PAR1 ICL2 peptide containing the YPX 3 L motif was immobilized on streptavidin beads and incubated with GST, GST–ALIX V domain, GST–ALIX V F676D, or the GST–ALIX Bro1 domain. Pulldowns (PD) were analyzed for the presence of bound protein by immunoblotting. Input was analyzed by Coomassie staining.

Article Snippet: GST vector, GST–ALIX V, GST–ALIX V F676D, and GST-ALIX Bro1 cDNA plasmids were transformed into BL21 (DE3) Escherichia coli cells (Agilent Technologies).

Techniques: Binding Assay, Mutagenesis, Immunoprecipitation, Western Blot, Transfection, Incubation, Staining