RNAi-mediated knockdown of <i>AjAPN1</i> transcript and its encoded protein.

<p>Third instar larvae were intrahemocoelically injected with target and control gene siRNA duplexes at dose of 5 µg/100 mg body weight followed by analyses of target gene/protein expression level at different time points. Observations obtained at 66 h post-injection are represented. Values represented are mean±standard deviation of three independent experiments (n = 3). Significance between groups was tested by One-Way ANOVA followed by Student-Newman-Keuls’ (SNK) test using SigmaPlot 11.0 software. *indicate statistical significance (P<0.05). Control (Cont): double-stranded <i>GFP</i> siRNA injected and Experimental (Expt): double-stranded <i>AjAPN1</i> siRNA injected insects. (A) Real-time quantitative PCR analysis. 18S rRNA was used as an internal endogenous control. Note that the fold decrease in <i>AjAPN1</i> transcript level in fat body and Malpighian tubule was 1.9 and 2.8 respectively. Semi-quantitative analysis is represented by the gel images. Here, <i>β</i>-actin gene was used as an internal endogenous control (lower panel). (B) Western blot analysis. Note the substantial reduction in the 113 kDa AjAPN1 protein band of fat body and Malpighian tubule of the target gene siRNA injected insects. β-actin expression was used as an internal endogenous control (lower panel). (C) APN activity analysis. Note the significant decrease in the APN activity level of fat body and Malpighian tubule of the target gene siRNA injected insects. Fb: fat body, Mt: Malpighian tubule and Sg: salivary gland.