Fig. 2

DCTN1 knockdown leads to the formation of ubiquitin-positive inclusions of TDP-43 in Drosophila. a Confocal microscopic images of TDP-43 antibody staining of larval eye discs of TDP-43 flies coexpressing EGFP-IR or dDCTN1-IR (#1 or #2). Arrows indicate cytoplasmic inclusions of TDP-43. Hoechst 33342 was used for nuclear staining. Scale bars, 20 µm (left) and 10 μm (right). The panels on the right are magnified images of the boxed region in the left panel. b Bar graph of the ratio of cells with cytoplasmic inclusions of TDP-43 shown in a. Data are presented as the mean ± SEM of 7–10 fly larvae. Statistical analysis was performed to assess differences from the EGFP-IR-expressing TDP-43 flies by one-way ANOVA followed by the Dunnett multiple comparison test (****p < 0.0001). c Confocal microscopic images of TDP-43 and ubiquitin antibody staining of larval eye discs of TDP-43 flies coexpressing EGFP-IR or dDCTN1-IR#2. Hoechst 33342 was used for nuclear staining. Scale bar, 5 μm. Fly genotypes: TDP-43/EGFP-IR, GMR-Gal4/+;;TDP-43/EGFP-IR; TDP-43/dDCTN1-IR#1, GMR-Gal4/+;dDCTN1-IR#1/+;TDP-43/+; TDP-43/dDCTN1-IR#2, GMR-Gal4/dDCTN1-IR#2;;TDP-43/+. d Western blot analysis of head lysates using antibodies against TDP-43. Smear bands with a molecular weight of more than 75 kDa were detected, and increased in the flies coexpressing dDCTN1-IR. e Schematic representation of the fractionation of fly head lysates using Triton X-100 and sarkosyl buffers. f Immunoblot images of fly head lysates fractionated by detergents. Fly genotypes: +, GMR-Gal4/+; TDP-43, GMR-Gal4/+;;TDP-43/+; TDP-43/dDCTN1-IR, GMR-Gal4/+;dDCTN1-IR#1/+;TDP-43/+. g, h Autocorrelation curves (g) and bar graph showing correlation time (h) obtained from FCS analysis of head lysates of TDP-43 flies. Fly genotypes: TDP-43, GMR-Gal4/+ ;TDP-43-EGFP/+ ; TDP-43/dDCTN1-IR, GMR-Gal4/+;dDCTN1-IR#1/+;TDP-43-EGFP/+