Journal: Nucleic Acids Research

Article Title: Tyrosine kinase c-Abl couples RNA polymerase II transcription to DNA double-strand breaks

doi: 10.1093/nar/gkz024

Figure Lengend Snippet: Formation and p-Dicer-dependent turnover of dsRNA at DSBs. ( A ) ChIP analysis of GFP-RNaseH1 occupancy at DS1 using site-specific primers. ( B ) Quantitative real-time PCR (qRT-PCT) of DNA immunopurified from DNA–RNA hybrids (DRIP) or upon incubation with recombinant RNaseH at DS1 using S9.6 hybridoma supernatant and region-specific primers. ( C ) qRT-PCR analysis of transcripts associated with CTD Y1P and immunopurification (mNET-IP). Values were normalized to data in absence of GFP-RNaseH1. (A–E) Asterisk, P -value < 0.05, two-tailed t -test. Error bar: mean ± SEM, n = 3. ( D, E ) Imaging and RGB quantitation of CTD Y1P and p-Dicer (p-DCR-1). White box, 2.5× zoom; n , number of cells with shown phenotype in %. ( F ) qRT-PCR of cDNA after J2 immunoselection with (J2 RIP) and reverse transcription with forward (upper panel)- and reverse (lower panel)-oriented primers spanning a region up to 1000 nts distant from DS1 in presence (J2+) or absence (J2-) of 4OHT, or upon preincubation of lysed material with recombinant RNaseIII (J2+ RNaseIII) prior to J2 RIP. J2+ values were set to 1. Asterisk, p-value <0.05, two-tailed t-test. Error bar: mean ±SEM, n = 3. ( G ) Autoradiograph detecting J2 immuno-selected (RNA IP) or total (IN) RNA or pBR322 MspI digest (M) upon end-labeling and PAGE separation. AU, arbitrary units. ( H ) as in (D), but preincubated with Leptomycin B (LMB) and stained with J2. Representative images are shown.

Article Snippet: Cells were permeabilized with PBS/0.3% Tween-20 (10 min, RT), washed 1× in PBS and incubated for 20 min at RT with either BSA (Sigma, 0.2 μg/ml final conc., diluted in PBS containing 0.02 mM NaOAc and 0.2 mM Tris), RNaseA (Sigma, 0.2 μg/ml final conc., diluted in PBS containing 0.02 mM NaOAc and 0.2 mM Tris) or RNaseIII (NEB, 2U final conc., diluted in RNase-free H 2 O containing 1× commercial reaction buffer (NEB) prior to fixation.

Techniques: Real-time Polymerase Chain Reaction, Incubation, Recombinant, Quantitative RT-PCR, Immu-Puri, Two Tailed Test, Imaging, Quantitation Assay, Autoradiography, End Labeling, Staining