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. 2012 Dec 27;7(12):e53256. doi: 10.1371/journal.pone.0053256

Figure 4. Met RNAi disrupts the 20E-triggered transcriptional cascade.

Figure 4

RNAi was performed during initiation of the early wandering stage (A–C) and ∼6 hr after pupation (D). The RNAi knockdown efficiency by Met2 RNAi is higher than for Met1 RNAi, and the downregulation rate of Met1 by Met2 RNAi is higher than for Met2 by Met1 RNAi. The #1 set of Met dsRNA was used. egfp dsRNA was used as a control. (A) Met1 and Met2, and the 20E-response genes EcR, USP, E74A and Br-C, as determined by qPCR, were significantly downregulated 24 hr after Met RNAi. RpL23 is used as a negative control of 20E-response gene. (B) MET1, USP and Br-C protein levels, as determined by Western blots, significantly decreased 24 hr after Met RNAi. Tubulin was used as a loading control. (C) USP and Br-C protein levels, as estimated by immunohistochemistry, significantly decreased 24 hr after Met RNAi. Localization of USP (green) and Br-C (red) were restricted to nuclei (Bar: 50 µm). (D) RNAi was performed ∼6 hr after pupation. The rest is as in (A).