Figure 2: Degradation of NMD substrates is traceable by xrFrag analysis.
From: Interrogating the degradation pathways of unstable mRNAs with XRN1-resistant sequences
(a) Depiction of the β-globin reporter mRNAs as in Fig. 1. (b) Northern blot of RNA samples extracted from HeLa cells transfected with the indicated siRNAs and reporter constructs. Co-transfected LacZ served as control mRNA. Mean values of reporter and xrFrag signal±s.d. (n=3) were quantified and for each knockdown condition the PTC values were normalized to the WT. The ratio of xrFrag to reporter mRNA levels is indicated below the graph. (c) Western blot analysis of siRNA knockdown efficiency using the indicated antibodies. Tubulin served as loading control. (d–f) Total RNA was extracted from stable HeLa Flp-In T-REx cells expressing the indicated reporter RNA and analysed by northern blotting. (e,f) The cells were transfected with the indicated siRNA 72 h before induction of expression. 7SL RNA served as endogenous control RNA. Unless indicated otherwise (f), reporter mRNA expression was induced for 24 h with 1 μg ml−1 doxycycline (Dox). Mean values of reporter and xrFrag signal±s.d. (n=3) were quantified and normalized to the WT control (+Dox for d; Luc or UPF1 knockdown for e; 12 h after Dox for f). The ratio of xrFrag to reporter mRNA levels is indicated below the graph.
