Supplementary Figure 4: TBK1 expression in patient-derived LCLs, fibroblasts and keratinocytes at mRNA and protein level.
From: Haploinsufficiency of TBK1 causes familial ALS and fronto-temporal dementia
(a) qRT-PCR measurement of TBK1 mRNA abundance in LCLs, fibroblasts and keratinocytes of 3-5 healthy controls and respective TBK1 mutant cell lines normalized to TBP. The bars indicate mean ± s.d. (b) PCR products amplified from LCL-derived cDNA using oligonucleotides spanning Exon 3 and 5 of TBK1 mRNA separated on a 1% agarose gel. The arrowhead marks a band exclusively observed in LCLs carrying the direct TBK1 splice site mutation c.358+2T>C. Sequencing of this band reveals the retention of the first 226 bp of the Intron separating Exon 4 and 5 in the mature mRNA and usage of an alternative splice site. The molecular size marker is indicated in bp. (c) PCR products amplified from fibroblast-derived cDNA using oligonucleotides spanning Exon 17/18 and 21 of TBK1 mRNA separated on a 1% agarose gel. The arrowhead marks a band exclusively observed in fibroblasts carrying the TBK1 c.2138+2T>C mutation. Sequencing of this band reveals the predicted skipping of Exon 20 in the mature mRNA. The molecular size marker is indicated in bp. (b, c) are representative images out of 2 replicates. (d, e) Western blots comparing TBK1 protein expression of LCLs (d) and fibroblasts (e) from healthy controls and respective TBK1 mutant cell lines. Western blots were repeated three times The arrowhead in (e) marks a band corresponding to the expression of a truncated TBK1 protein missing amino acids 690-713. ß-Actin is shown as a loading control. Molecular weights are indicated in kDa (All TBK1- and Actin-bands in (d) result from the same Western blot but were re-arranged for better presentation).
