Fig. 3: K136 mutations alter the RNA binding and splicing capabilities of TDP-43.

a HEK293E sh^TDP-43 cells were transfected with an empty plasmid, wt or K136Q TDP-43. The lysates were then incubated with biotinylated (UC)₁₂ or (UG)₁₂ RNA oligomers. Protein-RNA mix was incubated with magnetic streptavidin beads. Bound proteins were eluted and analyzed by Western blotting. N = 3 biological replicates. Source data are provided as a Source Data file. b Increasing concentrations of 6xHis purified TDP-43 proteins were incubated with biotinylated (UG)₁₂ oligomers. The resulting complexes were analyzed by a filter-binding assay. Membranes were incubated with HRP-coupled streptavidin. One of the [K136Q]TDP-43 data points illustrates overload problems at high protein concentration (>875 nM), precluding firm establishment of saturation binding curves. Source data are provided as a Source Data file. c Quantification of three replicates of filter-binding assay of wt (full line) and K136Q (dashed line) TDP-43 to biotinylated (UG)₁₂ RNA oligomers. **p < 0.01. Data are presented as mean values ± SD. Unpaired, two-sided t-test was used for comparison. Source data are provided as a Source Data file. d Representative Western blot of the protein levels before and after the purification of native TDP-43 via NiNTA pulldown. N = 3 biological replicates. Source data are provided as a Source Data file. e HEK293E sh^TDP-43 cells were cotransfected with the specified TDP-43 constructs and a plasmid containing the CFTR minigene. RNA was extracted and splicing of CFTR exon 9 was assessed via rtPCR (upper panel). Protein levels are shown in the lower panel. N = 3 biologically independent samples. f Quantification of three replicates assessing effects of mutations at different lysines of TDP-43 on the splicing of CFTR exon 9. Data are presented as mean values ± SD. Unpaired, two-sided t-test was used for comparison. **p < 0.01. Source data are provided as a Source Data file.