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Fig. 3. DmCdc6 RNA interference. (A) The regions of the DmCdc6 gene used for RNAi and RT-PCR in all experiments presented in the paper. DmCdc6 RNAi 1 and 2 were used in the standard Dmcdc6 knockouts presented in Fig. 3, and RT-PCR 1 is used in both this Figure and Fig. 7 to check for the presence of bulk DmCdc6. UTR, RNAi and RTPCR2 are used to knockout and check for, respectively, the presence of the endogenous DmCdc6 in the experiments discussed in Fig. 7. (B) Western blot analysis of Cdc6 depletion by RNAi: total cell extracts of 500,000 cells untreated (–) or treated with DmCdc6 double-stranded RNA (+) at day 3 and day 5 of the experiment were loaded in each well. (C) Depletion of DmCdc6 mRNA detected by RT-PCR at day 3 and day 5. –, no treatment; +, treated with Dm Cdc6 double-stranded RNA and m, treated with unrelated double-stranded RNA. The Df31 gene amplification serves as a nonspecific control and the ORC1 gene amplification serves as a specific control to test for cross reaction of the probes with the DmORC1 gene. (D) Comparison of the growth of untreated S2 cells, cells treated with DmCdc6 double-stranded RNA and cells treated with an unrelated double-stranded RNA. (E) FACS analysis of S2 cells treated with DmCdc6 RNAi compared with mock treated cells and cells treated with RNAi against cyclin E.
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