Fig. 2.

Knockdown of BRWD3 but not of JET inhibits light-dependent degradation of dCRY in S2 cells. A S2 cell line expressing dCRY-V5H under the Ac5 promoter was used. The S2-dCRY-V5H cells were treated with 1 μg of dsRNAs every other day four times and exposed to light for the indicated times. Then, aliquots were analyzed for Jet and Brwd3 expression by RT-PCR and for dCRY by immunoblotting. (A) Knockdown of Jet and Brwd3. Aliquots from dsRNA-treated cells were used to analyze the knockdown of Brwd3 and Jet mRNA by quantitative real-time PCR and normalized to Gapdh expression. Error bars represent SEM of three independent experiments. (B) Light-induced proteolysis of dCRY-V5H following Jet and Brwd3 (CDS and UTR) down-regulation analyzed by immunoblotting. Actin was used as a loading control. (C) Quantitative analysis of three dCRY proteolysis experiments including the one shown in B. Error bars represent the SEM of three independent experiments.