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. 2012 Jul 18;40(18):9255–9271. doi: 10.1093/nar/gks662

Figure 8.

Figure 8.

End-labeled RNAs detected by co-immunoprecipitation with hAgo1 antibodies. (A) Denaturing PAGE analysis of SG224 L-sshRNAs isolated from Ago1 antibody-bound complexes that were either 5′- or 3′-end-labeled as marked prior to transfection in 293FT cells (lanes 5′-SG224-Ago1 and 3′-SG224-Ago1, respectively) (B) Denaturing PAGE of the supernatant fraction of L-sshRNAs not immunoprecipitated by hAgo1 antibody (5′-SG224-Ago-UB and 3′-SG224-Ago1-UB, respectively). Full-length 5′ and 3′-end-labeled shRNAs [lanes in (A) and (B) labeled 5′-SG224 and 3′-SG224] were loaded as markers alongside the RNA that co-immunoprecipitated with hAgo1]. An arrow points to the immunoprecipitated SG224 RNA whose length is consistent with cleavage of the passenger arm at the slicer site. (C) Comparison of 3′-end-labeled R-sshRNAs isolated from Ago1 antibody-bound complexes that are active (lanes SG68-Ago1 and SG150-Ago1) with an R-sshRNA containing mismatches at the slicer site with reduced activity (SG148-Ago1) as well as the RNA fraction remaining unbound to hAgo1 antibodies (D). PS (passenger strand sliced), GS (guide strand sliced), LC (loop cleaved).