Fig 2: Lysine 95 is critical for the inhibition of nuclear import of TDP-43 by poly-GA ADomain structure of TDP-43 and location of the bipartite NLS at positions 78–99 (Winton et al, 2008). Known ubiquitination sites listed on http://www.phosphosite.org at K84 and K95 are highlighted.BImmunoblot of HeLa cells transfected with RFP-based TDP-NLS wild type (WT) or mutants (K84A, K84R, K95A, K95R).C, DHeLa cells were co-transfected with the indicated TDP-NLS reporters as well as GFP or GA175-GFP, and treated with MG132 (10 µM) or vehicle for 16 h. (D) Automated quantification of RFP-NLS reporters in GFP-positive cells. Note that K84A and K84R block overall import, while K95A and K95R allow import but are resistant to inhibition by poly-GA. n = 4 biological replicates. The total number of cells analyzed per group was (from left to right) 667, 581, 789, 783, 809, 708, 628, 721, 938, 557, 857, 861, 886, 699,789, 539, 636, 577, 638, and 870. Scatter plot with bar graphs of mean ± SD. One-way ANOVA with Tukey's multiple comparisons test. ***P < 0.001. Scale bar denotes 20 µm.E, FHeLa cells were co-transfected with the indicated GFP-TDP-NLS reporters and iRFP or GA175-iRFP. Cell lysates were immunoprecipitated with anti-GFP and immunoblotted with indicated antibodies to detect co-immunoprecipitation of the TDP-43 NLS with importin-a5/KPNA1 nuclear import receptor. (F) Quantification of KPNA1 levels normalized to total GFP-NLSTDP reporter levels in anti-GFP immunoprecipitates. n = 3 biological replicates. Scatter plot with mean ± SD. One-way ANOVA with Tukey's multiple comparisons test. ***P < 0.001. See also Fig EV4. Red dashed line indicates the control's expression level. Source data are available online for this figure.