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. 2012 Oct 16;7(10):e47409. doi: 10.1371/journal.pone.0047409

Figure 1. Validating the candidate genes obtained via 2D DIGE and RISC IPs.

Figure 1

A. Measuring levels of candidate transcripts obtained by RISC IPs in Ago2-immunoprecipitated RNA samples of BJAB BARTs and BJAB Empty cell lines by RT-PCR revealed an enrichment for IPO7 transcripts in immunoprecipitates of BJAB BARTs cells (*indicates p-value <0.0005, Student’s t-test). B. Reporter constructs containing the 3′UTRs of candidate genes were transfected into BJAB BARTs and BJAB Empty cells. The normalized luciferase activity in BJAB Empty cells was arbitrarily set to 100% (*indicates p-value <0.05, Student’s t-test). The luciferase activity of IPO7 3′UTR-pmiRGLO construct in BJAB BARTs cells decreased by 33%. C. Expression levels of CEBPA, PUMA, CNBP1 and IPO7 in BJAB Empty and BJAB BARTs cells were analyzed by immunoblotting, quantified by Image J software and normalized to levels of alpha-tubulin. The levels of expression of all proteins are indicated under the respective bands. The levels of IPO7 protein were found to decrease by 33% in the presence of BART miRNAs in three biological replicates while the levels of the other three proteins were unaffected by them. D. Nine candidates identified either by 2D DIGE or RISC IPs were examined further either by RT-PCR to measure their levels in RISCs, in reporter assays, or by immunoblotting where appropriate antibodies were available. IPO7 was validated by all three methods.