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. 2020 Nov 6;143(12):3827–3841. doi: 10.1093/brain/awaa317

Figure 5.

Figure 5

Impact of both TARDBP variants on the splicing regulation functionality of TDP-43, demonstrated by splice-in/out add-back assay of CFTR exon 9. From left to right: The first two lanes show the baseline measurement with both splicing in and out of exon 9 in the absence (-) or presence of TDP-43 siRNA (+). Lane 3 shows that addition of si-resistant wild-type TDP-43 can rescue the splicing functionality (WT) but this cannot be achieved by a TDP-43 carrying the F4L mutation that does not allow the protein to bind RNA (lane 4). Lanes 5 and 6 show the results obtained after the addition of mutated TDP-43 carrying the predicted damaging variants (R42H and L41F). Middle: Western blots showing equal expression of the flagged-TDP-43 wild-type and mutants (pFlag-TDP-43s) following knockdown of the endogenous protein (end. TDP-43). Tubulin was used as an internal control. Bottom: Quantification of the ratio of CFTR exon 9 inclusion. The standard deviation and P-values are reported for three independent experiments. Unpaired t-test was performed for statistical analysis (*P < 0.05).