Figure 3.
Titration of limiting Ago proteins by exogenous siRNA. (A) Developmental control of accumulation of xlAgo proteins in early embryos. Western blots of whole-cell extracts from eggs or embryos were probed with antibodies specific for the indicated proteins (cf. Supplemental Fig. S2); two (left panels) or one (right panels) embryo equivalents were loaded per lane. (B) Expression of exogenous Ago or TRBP proteins in early embryos. One-cell embryos were injected with siRNANS (30 fmol per embryo) alone or together with in vitro synthesized mRNAs encoding Myc-tagged human proteins, as indicated, and protein levels at stage 9.5 were monitored as in A. Note that the α-Ago antibody (anti-EIF2C2, Abnova) detects all three hAgo proteins, albeit with different efficiencies (cf. α-Ago and α-Myc signal). Kap-β and xlExp5 served as loading controls in A and B, respectively. (C) Suppression of siRNANS-mediated inhibition of miR-427 processing by exogenous Ago proteins. Accumulation of miR-427 in embryos from the experiment shown in B was monitored by Northern blot analyses as in Figure 2A. (D) Limiting Ago proteins in early embryos. Embryos were programmed to express wild-type (wt) or catalytically inactive (mut) Myc-hAgo2 in the presence of siRNANS (20 fmol per embryo), and miR-427 biogenesis was monitored as in C. (E) PhosphorImager quantification of mature miR-427 hybridization signals in D.