hnRNP K induces the protein expression of endogenous TDP-MP20. (A) Immunocytochemistry of HeLaS3 cells overexpressing FLAG-hnRNPs. MP20-endo (green), FLAG (magenta), and Hoechst (blue). Yellow boxes indicate magnified images (scale bar: 10 μm). Arrowheads and arrows denote FLAG-positive and FLAG-negative cells, respectively. Scale bar: 50 μm. (B) Quantification of nuclear MP20 fluorescence intensity in FLAG-hnRNPs–positive cells. A total of 100 FLAG-positive and 100 FLAG-negative cells per group were analyzed. Fluorescence signals were normalized to the average intensity of 400 FLAG-negative cells. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s test. (C) Comparison of nuclear MP20 signals in cells with FLAG-hnRNP K localized in the nucleus only (Nuc) versus in both the cytoplasm and nucleus (Cyto+Nuc). Nuc: 123 cells; Cyto+Nuc: 177 cells. Welch’s t test was used for statistical analysis. (D and E) WB analysis of HEK293T cells overexpressing FLAG-hnRNPs (D) or with hnRNP K KD (E). Quantification of the relative expression of FL-endo, SVs-endo, and MP20-endo (n = 3 for each group). Data are normalized to β-actin. Statistical analyses were performed using one-way ANOVA followed by Dunnett’s test (D) or Welch’s t test (E). (F) IP of FLAG-hnRNP K overexpressed in HEK293T cells using MP20 or FLAG Ab, with or without peptide absorption by MP20- or MP18-immunizing peptides. An anti-IgG Ab was used as a negative control. All graphs show the mean ± SEM. *P < 0.05, **P < 0.01, and ****P < 0.0001. Ab, antibody. Source data are available for this figure: SourceData F5.