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Fig. 3 | BMC Biology

Fig. 3

From: TDP-43 prevents retrotransposon activation in the Drosophila motor system through regulation of Dicer-2 activity

Fig. 3

TBPH physically interacts and influences Dcr-2 levels. a Western blot analysis of lane 1 (+/+;D42-GAL4,UAS-EGFP/+), lane 2 (+/+;D42-GAL4,UAS-EGFP/UAS-GFP-IR), lane 3 (tbphΔ23/tbphΔ23; D42-GAL4,UAS-EGFP/+), and lane 4 (tbphΔ23/tbphΔ23; D42-GAL4,UAS-EGFP/UAS-GFP-IR). Adult brains, 1 day old, were probed with anti-GFP and alpha-tubulin antibodies. The same membrane was probed with the two antibodies, and the bands of interest were cropped. Quantification of normalized amounts was reported below each lane. n = 2 (independent biological replicates). b Real-time PCR of Dcr-2 transcript levels normalized on Rpl11 (housekeeping) in adult heads of w1118 and tbphΔ23/tbphΔ23. n = 3 (independent biological replicates, with 2 technical replicates for each), *p < 0.05, calculated by two-tailed t test, error bars SEM. c Western blot analysis of w1118 and tbphΔ23/tbphΔ23 adult brains probed with anti-Dicer and anti-actin antibodies. The same membrane was probed with the two antibodies, and the bands of interest were cropped. Quantification of normalized amounts and SD was reported below each lane. n = 4 (independent biological replicates), *p < 0.05, calculated by two-tailed t test. d Western blot analysis on human neuroblastoma (SH-SY5Y) cell line probed for anti-Dicer, anti-GAPDH, and anti-TDP-43 in siGFP (GFP ctrl) and siTDP-43 (TDP-43 silenced). The same membrane was probed with the three antibodies, and the bands of interest were cropped. Quantification of normalized protein amount and SD was reported below each lane. n = 3 (independent biological replicates), **p < 0.01, calculated by two-tailed t test. e qRT-PCR analysis of mRNAs immunoprecipitated by FLAG-tagged TBPH (Elav>TBPH) and its mutant variants TBPH-RBDmut (Elav>TBPH-RBDmut). The dicer-2 enrichment folds were referred to as rpl-11 (negative control), and syntaxin has been used as a positive control. n = 2 (biological replicates). f Western blot analysis of TBPH (GMR-GAL4/UAS-TBPH) and + (GMR-GAL4/+). Input, depleted, and immunoprecipitated (IP) materials were analyzed, probing the membrane with anti-TBPH, anti-Dicer, and anti-GAPDH. n = 4 (biological replicates). Individual data values are provided in Additional file 6: Individual Data Values.pdf

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