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. 2008 Dec 1;22(23):3292–3307. doi: 10.1101/gad.1734608

Figure 3.

Figure 3.

Physical interaction of UNC-51, UNC-76, and KHC. (A) Myc-tagged UNC51 (full-length) and HA-tagged UNC-76 (truncation mutants) were coexpressed in HEK293T cells as indicated. Cell lysates were immunoprecipitated by anti-HA followed by immunoblot with anti-myc. (*) Nonspecific band (22 kDa) detected by anti-HA antibody. (B) UNC-51 binds UNC-76 in vitro. GST alone or GST fused with full-length UNC-76 was produced in E. coli, purified on Glutathione-Sepharose beads, and incubated with myc-tagged UNC-51 C′-terminal domain (amino acids 557–855) produced by in vitro translation (IVT). Proteins that bound to the beads were eluted and analyzed by immunoblot using anti-myc. (C) Schematic representation of the domains involved in UNC-51−UNC-76 binding. Black bars show deletion constructs that bind the other in coimmunoprecipitation assays. Constructs shown in gray did not bind. Binding of all deletion constructs except for UNC-76 (118−340 amino acids) were tested by coimmunoprecipitation assays in A and Supplemental Figure S5, whereas no binding of UNC-76 (118−340 amino acids) with UNC-51 was confirmed by GST pull-down assay (data not shown). The UNC-76 and UNC-51 functional domains are indicated within each protein. (D) UNC-51 forms a complex with KHC via UNC-76. HEK293T cell extracts containing heterologously expressed UNC-51 (full-length), UNC-76 (full-length), and KHC (full-length), as indicated, were analyzed by immunoprecipitation with anti-HA followed by immunoblot with the indicated antibodies.