Figure 1

Knockdown of UPF1, UPF2 and SMG1 results in the production of GFP protein by cells stably expressing pGFPint. A Upper panel: western blot of 10μg total cell protein for UPF1 and actin as a loading control. Lower panel: western blot of 30 μg total cell protein for UPF2 and actin as a loading control. In each case the C2 treated sample was diluted 1:2, 1:4 and 1:8 in RIPA buffer in order to better estimate the degree of knockdown in the knockdown sample. Representative samples of three biological repeats are shown. B. Histogram comparing the fold change in SMG1 mRNA levels in response to treatment with C2 (black bar) or SMG1_A (light grey bar) siRNAs, as measured by QPCR on parallel RNA samples. The height of each bar represents the mean of three biological repeats, while error bars represent the standard error of the mean (SEM). C. Western blot of 10 μg total cell protein for GFP and actin as a loading control. Representative samples of three biological repeats for each siRNA are shown. D. Histogram comparing the fold change in mRNA levels for GFP and the NMD sensitive isoforms of SC35 (1.6 kb and 1.7 kb, [36]) in response to treatment with each siRNA, as measured by QPCR on parallel RNA samples. The height of each bar represents the mean of three biological repeats, while error bars represent SEM. The colour scheme is indicated in the side panel. E. Schematic of AS-NMD events within the 3' UTR of SC35 (SFRS2). Dark boxes represent exons, and white boxes introns. Dashed lines denote alternative splicing patterns and arrows denote the QPCR primers used in D.