Figure 6.

Cycloheximide increases abundance of CLK1 PTC⁺ isoform. (a) Gene structures of CLK1 full-length and PTC+ isoforms as determined by our analysis. (b) Menegay et al. [48] performed the RT-PCR analysis of CLK1 isoforms; Figure 8 of that analysis [48] is reproduced here with permission (^© Company of Biologists Ltd.). The 560 bp fragment corresponds to the full-length CLK1 isoform; the 453 bp fragment corresponds to the PTC⁺ CLK1 isoform. The analysis shows that cycloheximide, but not UV irradiation or high salt (data not shown), increased the relative abundance of the CLK1 isoform containing a premature termination codon. As cycloheximide is a potent inhibiter of NMD (see, for example, [28,51-53]), this result suggests that the CLK1 PTC⁺ isoform is degraded by NMD. Menegay et al. [48] describe their figure as follows: "Shift in PCR products of splice forms with cycloheximide. Control or PC12 cells treated with 10 μg/ml cycloheximide for 60 minutes were harvested, RNA was extracted, and RT-PCR was performed. [...] PCR products of the 560 bp full-length form or the 453 kinase-less form of CLK1 message shown. [...] PCR of GAPDH controls from each sample to control for RNA loading."