Precipitation of mRNAs by Ago2 and phosphorylation mutants. Homo sapiens

Argonaute (Ago) proteins associate with microRNAs (miRNAs), which guide them to complementary target mRNAs resulting in gene silencing. RNA coimmunoprecipitations (RIP) were performed with Ago2 and Ago2 mutants. The precipitated mRNAs were analyzed using one Affymetrix microarrays to compare the mRNA preciptation rates of mutants vs Ago2 wild type. Overall design: HEK293 cells were transfected with plasmids expressing tagged h.s. Ago2 (wild type) or tagged h.s. Ago2 mutants Ago2_824-34A (Phos3A), Ago2_824-34E(Phos3E) and Ago2-Y529E(529E). Following RIP, mRNA was extracted and hybridized to Affymetrix microarrays. The Input Samples and the IP samples underwent separate VSN ERCC normalization.