NLRC5 requires an intact nuclear localization signal for efficient MHC class I induction.
(A) Sequence of the NLRC5 bipartite NLS in the N-terminus and the indicated mutant versions used in this study. Alanine substitution of the right or left arm of the NLS was used to construct the NLSI and NLSII import mutant expression plasmids. DM: double mutant. (B–F) HEK293T cells were transiently transfected with either expression vectors for GFP, GFP-NLRC5, or GFP-CIITA. (B) Subcellular localization of wild-type and NLS mutant NLRC5 upon LMB treatment (100 nM, for 2 hrs). Scale bar: 10 μm. (C) Reporter gene analysis of the HLA-B promoter. An HLA-B luciferase reporter construct was co-transfected with the indicated plasmids and cell lysates were analyzed 48 hrs post transfection by dual-luciferase assay. Data are a representative of several independent experiments performed in duplicates and are plotted as fold induction with respect to the GFP control vector (--). Error bars represent ± SD. *p < 0.05. (D) Cell extracts were examined for the expression of MHC class I heavy chain by immunoblotting 48 hrs post transfection. Sh. Exp., short exposure. MHC class I signal intensities, obtained by densitometric analysis, were normalized by β-Actin levels (bar diagram). The GFP blot demonstrates equal expression of the NLRC5 and CIITA fusion proteins. (E) RNA samples were isolated and HLA-B transcripts were quantified by qRT-PCR using gene specific primers. (F) Quantitative analysis by flow cytometry using an anti-MHC class I antibody. WT (black line), DM (grey line), empty vector control (shaded histogram).

An active NBD is required for nuclear translocation of NLRC5.
(A) Sequence of the changes introduced into the Walker A or B motifs of the NBD domain of NLRC5. (B) Subcellular localization of wild-type and NBD mutant NLRC5 upon LMB treatment (100 nM) as observed by spinning disc confocal microscopy. Cell nuclei were stained with Hoechst 33342 (blue). Scale bar: 10 μm. (C) Reporter gene analysis of HLA-B (upper panel) and HLA-DR promoters (lower panel), respectively. Luciferase reporter constructs were co-transfected with the indicated plasmids and cell lysates were analyzed 48 hrs post transfection by dual-luciferase assay. Data were plotted as fold induction with respect to the GFP control vector as the mean ± SD of duplicates. *p < 0.05.

The NBD is required for transactivation of MHC class I genes.
(A) Schematic representation of NLS-tagged NLRC5. (B–D) HEK293T cells were transiently transfected with the indicated expression vectors. (B) Subcellular localization of wild-type, NBD mutant or NLS tagged NLRC5 upon LMB treatment (100 nM, for 2 hrs) as observed by epifluorescence microscopy. Scale bar: 10 μm. (C) Quantified subcellular localization. Cells were counted in a blind manner as ‘cytosolic’ or ‘nuclear’ if the majority of the GFP signal was detected in the respective compartment, and ‘intermediate’ if the signal intensity in both compartments was comparable. Data was pooled from three independent experiments. Error bars represent ± SD. (D) Reporter gene analysis of the HLA-B promoter. An HLA-B luciferase reporter construct was co-transfectd with the indicated plasmids and cell lysates were analyzed 48 hrs post transfection by dual-luciferase assay. Data are a representative of several independent experiments performed in duplicates and are plotted as fold induction with respect to the GFP control vector (--). Error bars represent ± SD. *p < 0.05.