Characterization of dominant-negative TDP-43 isoforms. (A) Schematic representation of RT-PCR analysis for TDP-43–associated cryptic splicing targets (GPSM2 and ATG4B) and alternative splicing events (PDP1 and BCL2L11). Representative RT-PCR results from HEK293T cells overexpressing FLAG-MPs. TDP-FL KD was used as a positive control for CE inclusion and splicing exon inclusion (PDP1) or exclusion (BCL2L11). (B) RT-qPCR analysis of CE inclusion in GPSM2 and ATG4B transcripts in cells expressing FLAG-MPs. ACTB was used for normalization (n = 3 for each group). nd, not detected. (C) Ratios of exon inclusion to exon exclusion for PDP1 and BCL2L11 transcripts were determined via RT-PCR (n = 3 for each group) in cells expressing FLAG-MPs. (D) Schematic representation of TDP-FL, MP20, MP18, and their RRM domain mutants (Rmt). (E) Representative RT-PCR results of the TDP-43–associated splicing targets in HEK293T cells overexpressing FLAG-Rmt isoforms. (F) RT-qPCR analysis of CE inclusion in GPSM2 and ATG4B transcripts in cells expressing FLAG-Rmt isoforms. ACTB was used for normalization (n = 3 for each group). (G) Ratios of exon inclusion to exon exclusion for PDP1 and BCL2L11 transcripts in cells expressing FLAG-Rmt isoforms (n = 3 for each group). (H) Co-IP analysis of FLAG-MPs and TDP-FL fused to Venus (FL-Venus) in HEK293T cells. FLAG IP was performed, and Venus levels were normalized to FLAG signals (n = 3 for each group). (I) Proposed model of dominant-negative effects exerted by TDP-MP20 on splicing regulation. In B and C, data were analyzed using one-way ANOVA followed by Dunnett’s test. In F, G, and H, data were analyzed using one-way ANOVA followed by Tukey’s test. Error bars represent mean ± SEM. *P < 0.05, **P < 0.01, and ****P < 0.0001. Gly-rich, glycine-rich region; NLS, nuclear localization signal. Source data are available for this figure: SourceData F2.
Figure 2.

Characterization of dominant-negative TDP-43 isoforms. (A) Schematic representation of RT-PCR analysis for TDP-43–associated cryptic splicing targets (GPSM2 and ATG4B) and alternative splicing events (PDP1 and BCL2L11). Representative RT-PCR results from HEK293T cells overexpressing FLAG-MPs. TDP-FL KD was used as a positive control for CE inclusion and splicing exon inclusion (PDP1) or exclusion (BCL2L11). (B) RT-qPCR analysis of CE inclusion in GPSM2 and ATG4B transcripts in cells expressing FLAG-MPs. ACTB was used for normalization (n = 3 for each group). nd, not detected. (C) Ratios of exon inclusion to exon exclusion for PDP1 and BCL2L11 transcripts were determined via RT-PCR (n = 3 for each group) in cells expressing FLAG-MPs. (D) Schematic representation of TDP-FL, MP20, MP18, and their RRM domain mutants (Rmt). (E) Representative RT-PCR results of the TDP-43–associated splicing targets in HEK293T cells overexpressing FLAG-Rmt isoforms. (F) RT-qPCR analysis of CE inclusion in GPSM2 and ATG4B transcripts in cells expressing FLAG-Rmt isoforms. ACTB was used for normalization (n = 3 for each group). (G) Ratios of exon inclusion to exon exclusion for PDP1 and BCL2L11 transcripts in cells expressing FLAG-Rmt isoforms (n = 3 for each group). (H) Co-IP analysis of FLAG-MPs and TDP-FL fused to Venus (FL-Venus) in HEK293T cells. FLAG IP was performed, and Venus levels were normalized to FLAG signals (n = 3 for each group). (I) Proposed model of dominant-negative effects exerted by TDP-MP20 on splicing regulation. In B and C, data were analyzed using one-way ANOVA followed by Dunnett’s test. In F, G, and H, data were analyzed using one-way ANOVA followed by Tukey’s test. Error bars represent mean ± SEM. *P < 0.05, **P < 0.01, and ****P < 0.0001. Gly-rich, glycine-rich region; NLS, nuclear localization signal. Source data are available for this figure: SourceData F2.

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