Journal: PLoS ONE

Article Title: Cbp80 is needed for the expression of piRNA components and piRNAs

doi: 10.1371/journal.pone.0181743

Figure Lengend Snippet: (A, D) Knock down of Cbp80 in egg chambers expressing a dsRNA against Cbp80 (dsCbp80 ) driven by the GAL4-nos.NGT40 driver and the UAS-Dcr2. Control flies expressed a dsRNA against GFP ( dsGFP ) (B, C) Ovaries expressing specifically in the germline ( pCog-Gal4 driver) shRNAs against Cbp80 or mCherry (as control). Control flies used for RT-PCR experiments expressed also a Jupiter-mCherry fusion protein. (A-B) mRNA levels of piRNA pathway factors ( piwi , aub , Rhi , zuc and Ago3 ) and Cbp80 were measured by qRT-PCR. Fold expression levels of each mRNA in the knockdown samples relative to its expression in the corresponding control are shown for each mRNA. Total starting RNA amounts were the same in both samples. Error bars represent + /- SD of 2 controls in B and 3 control samples in A and 3 biological knockdown replicates. *p<0.05; **p<0.01; ***p<0.001. (C-D) Levels of Cbp80, Piwi, Aub, BicD, Cdk7, Clc and Tub (as loading control) were assessed by Western blotting. Ponceau staining is also shown to reveal total proteins as loading control. Levels Piwi, Aub and Cdk7 were strongly reduced upon Cbp80 reduction. On the other hand, levels of Tub, Clc and BicD were less affected. For Cbp80 knockdown samples were extracted from egg chambers showing the phenotype “d” .

Article Snippet: Western blotting was carried out with mouse anti-Piwi P3G11 (1:1,000; [ ]), mouse anti-Aub 4D10 (1:2,000; [ ]), rabbit anti-Cbp80 (1:1,000; [ ]), mouse anti-alpha tubulin primary antibodies (1:250; Developmental Studies Hybridoma Bank), mouse anti-BicD 1B11 (1:10; [ ]), mouse anti-Cdk7 (1:10 mix of 20H5 and 19E7.2 clones; [ ]) and rabbit anti-Clc (1:3000, [ ]).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Staining