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. 2017 Jan 19;38(3):297–309. doi: 10.1002/humu.23161

Figure 1.

Figure 1

Transcript and protein analysis of TBK1 LoF and single amino acid deletion mutations. A: gDNA and cDNA sequence traces around the c.288delT (p. Val97Phefs*2) mutation, showing reduced expression of the mutant transcript on cDNA extracted from blood. B: gDNA and cDNA sequence traces around the c.379C>T (p.Arg127*) mutation, showing the absence of the mutant transcript on cDNA extracted from blood. C: Sizing of cDNA fragments generated with primers in TBK1 exon 10 and exon 13 of the c.1340+1G>A (p.Ala417*) carrier on cDNA extracted from blood. Sequence traces from the low‐expressed aberrant transcript demonstrates skipping of exon 11. D: Transcript and protein analysis on brain frontal cortex from the c.235_237delACA (p.Thr79del) carrier and four age‐matched control brains. The graph on the left shows the relative expression in the patient sample (blue) compared with the control samples (black) measured by quantitative real‐time PCR (qRT‐PCR). In the middle, Western blot analysis is shown of protein extracts from the patient carrier compared with control individuals. The upper band represents TBK1 (84 kDa) and the lower band represents the housekeeping protein GAPDH (37 kDa). The graph on the right shows the quantification in the patient sample (blue) and control samples (black) of the TBK1 signal normalized to the signal of GAPDH. Error bars represent the SD. E: Western blot analysis of phosphorylated TBK1 (Ser172, p‐TBK1) (upper band, 84 kDa) in HEK293T cells overexpressing the in‐frame single amino acid deletions (p.Thr79del, p.Asp167del, and p.Glu643del) compared with wild type, relative to GAPDH (lower band, 37 kDa). Mock and kinase dead (p.Ser172Ala, KD) were used as negative control. cDNA numbering according to reference sequence NM_013254.3, in addition, for intronic variants, the genomic reference sequence NC_000012.12 was used. Nucleotide positions refer to cDNA sequence and nucleotide numbering uses +1 as the A of the ATG translation initiation codon in the reference sequence, with the initiation codon as codon 1. Protein numbering according to reference sequence NP_037386.1.