(A) Multi-species protein sequence alignment of residues in the UNC93B1 C-terminal tail. The YxxΦ motif, YRYL, is boxed. (B) Precursor TLR9 accumulates in UNC93B1-Y539A expressing cells. Lysates from 3d iMac cells expressing TLR9-HA were complemented with the indicated GFP tagged UNC93B1, analyzed by SDS-PAGE, and immunoblotted with anti-HA and anti-GFP antibodies. The precursor (black arrow), ER (white arrow) and cleaved (grey arrow) forms of TLR9-HA are indicated. n.s. indicates a non-specific band. (C) Mutations of YxxΦ in UNC93B1 confer partial phenotypes in TLR9 trafficking when compared with Y539A. Lysates from 3d iMac cells expressing TLR9-HA and GFP tagged UNC93B1-WT, H412R, Δ538, L542A, Y539F, Y539A were analyzed by SDS-PAGE and immunoblotted with anti-HA and anti-GFP antibodies. Presence of precursor (black arrow), ER (white arrow) and cleaved (grey arrow) forms of TLR9 are indicated. (D) TLR9 signaling is impaired in UNC93B1-Y539A cells. 3d iMac cells, complemented with GFP tagged UNC93B1-WT, -H412R, or -Y539A, were harvested for intracellular TNFα staining 5 hr after stimulation with 3 μM CpG, 1 μg/ml Pam3CSK4 (Pam) or left unstimulated. Percentages of TNF-producing cells after gating on UNC93B1-GFP positive cells are plotted. (E) The C-terminal tail of UNC93B1 interacts with AP-2μ. Results from a yeast two-hybrid assay testing for interaction between the AP (-1A, -2, -3A, -4) µ subunits and the N- or C-terminal cytosolic regions of UNC93B1 (N- or C-tail UNC), are shown. Growth on –His–Trp–Leu plates (–His) or –Ade–Trp–Leu (–Ade) indicates interaction. Growth on –Trp–Leu plates (+His+Ade) serves as a control. (F)–(G) Tyr-539 on UNC93B1 mediates interaction with AP-2 complex. (F) Results from a yeast two-hybrid assay testing for interaction between the AP-2µ subunit and the C-terminal cytosolic region of UNC93B1 (UNC93B1 C-tail) from WT, the Y539A mutant, or the Δ538 mutant are shown. Growth on –His–Trp–Leu plates (–His) indicates interaction. Growth on –Trp–Leu plates (+His) serves as a control. (G) HEK293T were transiently transfected with AP-2µ-HA and FLAG tagged UNC93B1-WT, -Y539A. Cell lysates were incubated with anti-FLAG matrix, and UNC93B1-associated proteins were eluted with FLAG peptide, separated by SDS-PAGE and visualized by immunoblot with anti-HA or anti-FLAG antibodies. UNC93B1 associated AP-2µ is indicated with black arrow. Results are representative of at least three experiments (B, D–F) or two experiments (C and G).