Unc93b cleavage does not affect TLR signaling or secretory pathway trafficking.

<p>(<b>a</b>) Schematic of wild-type Unc93b (Unc93b_Full), Unc93b-H412R mutant (Unc93b_H412R), Unc93b truncation mutants (Unc93bΔ17 or Un93bΔ27) and Unc93b cleavage fragments (Nterm₁₇ and Nterm₂₇). (<b>b, c</b>) 293T-FlagTLR3 (b) or HEK293XL-TLR8HA (c) cells were co-transfected with the indicated Unc93b construct containing a C-terminal Myc tag and/or N-terminal GFP tag, and IFNβ luciferase reporter (pIFNβ-fluc) and control renilla (pRL-null) constructs. Approximately 24 hours post transfection, cells were treated with 1 μg/mL poly(I:C) (b) or 10 μg/mL R848 (c) for 16 hours. Luciferase levels were analyzed using the Dual Luciferase reporter assay system. Firefly luciferase levels were normalized to renilla luciferase levels and then normalized to the vector transfected mock-treated condition. Data shown are an average of 2 independent experiments (b) or a representative experiment (c) and significance is compared to vector plus ligand. (<b>d</b>) Immunoblotting of lysates collected from 293T cells used in panel 4c and probed with antibodies against GFP and Myc (top) or GAPDH (bottom) as a loading control. (<b>e, f</b>) U2OS cells were co-transfected with Venus-fused VSV-G and the indicated Unc93b constructs containing a C-terminal Myc tag. Approximately 48–72 hours post-transfection, cells were fixed and then immunostained for Myc and confocal microscopy performed (<b>e)</b> and then images analyzed to obtain Pearson’s correlation coefficients (<b>f</b>).