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. 2009 Dec 9;38(5):1559–1574. doi: 10.1093/nar/gkp1115

Figure 7.

Figure 7.

Evidence that the pre-mRNA upregulatory response is independent of protein synthesis and UPF1. (A) Schematic diagram denoting the locations of the nonsense mutations in constructs AN1 and AN2 (also in Figure 1). (B, C) Quantification of RNase protection analysis performed on total cellular RNA (10 µg) harvested from HeLa cells incubated for 6 h with cycloheximide (CHX). Prior to CHX treatment, the cells were transiently transfected with the constructs shown, as well as a β-globin expression vector as an internal control, and cultured for 2 days. Probe 1 (A) was used to detect both the pre-mRNA and mature mRNA. The values shown are the average of two independent experiments that were normalized with β-globin mRNA, the internal control. Error bars indicate standard error. (D–F) Quantification of RNase protection analysis performed on total cellular RNA (10 µg) harvested from HeLa cells transiently transfected with a UFP1 siRNA to deplete UPF1 levels or a Luciferase (Luc) siRNA as a negative control. Probes 1 and 3 (A) were used to detect mature/IVS-A+ pre-mRNA and IVS-B+ pre-mRNA, respectively. Values were quantified from three independent experiments by the approach described in Figure 1.