Figure 8. Biochemical analyses of Uaa incorporation and NMD effect.

(A) Western blot analysis of JFFluc protein expressed in LWA717 animals grown on solid plates containing 4 mM or 0 mM OmeY. Animals were lysed, and the JFFluc protein was immunoprecipitated. Four elutions for each sample were collected and equal volumes were loaded into each lane. After transferring, the blot was probed with an anti-FLAG antibody to detect the purified JFFluc-3xFLAG. At the molecular weight corresponding to the JFFluc-3xFLAG protein (64 kDa) a strong band was visible for each elution of purified protein from animals grown on 4 mM OmeY. Only a weak band in the first 2 elutions was seen for animals grown in the absence of OmeY. Band densitometry revealed that in the absence of OmeY, the total protein detected was only 3.5% of that purified in the presence of 4 mM OmeY. (B) Fluorometric analysis of JFFluc proteins. The JFFluc protein purified from strain LWA718, which has JFFluc gene with a TAG codon at position 158, showed a fluorescence emission peak at 526 nm. The JFFluc protein purified from the control stain LWA1580 expressing the wild-type JFFluc gene (without the TAG158 codon) showed no detectable fluorescence. The same amount of protein from each strain was used for measurement. For comparison, the fluorescence emission spectra of the free Uaa DanAla and DanAla-Ala dipeptide were also measured, both of which had an emission peak at 538 nm. The dansyl fluorophore is sensitive to the environment, and the emission maximum changes after being incorporated into a protein. (C) Inactivation of NMD increased Uaa incorporation efficiency. LWA717 animals grown with 4 mM OmeY expressed more JFFluc protein when smg-1 was knocked down by RNAi (left lane), in comparison to the control RNAi against GFP (right lane). JFFluc protein was immunoprecipitated and detected by an anti-FLAG antibody in the Western. The mean fold change determined by band density when comparing smg-1 treated animals to the GFP control was 5.6, n=2.