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Figure 4 | BMC Molecular Biology

Figure 4

From: Clarifying mammalian RISC assembly in vitro

Figure 4

Endogenous pre-Let7a-3:Ago2 complexes guide 5'- and 3'- arm target cleavage in vitro. A. Endogenous pre-miRNPs were immunopurified using anti-Ago (2A8) antibody or from Dicer+/- or Dicer-/- mouse embryonic fibroblasts (MEFs). Non-immune mouse serum (NMS) was used as control. 30% of the total volume of agarose G beads from each immunoprecipitation (IP) was then incubated either with radiolabeled 5' -arm (Td-5) or with 3' -arm target (Td-3) and then treated with proteinase K. Predicted size of 5 -cleavage product of 5' -arm and 3' -arm target is 9 nt and 16 nt respectively. B and C. Expression of let-7a or let-7a * was detected by Northern blot of total RNA (20 μg/lane) isolated from Dicer+/- or Dicer-/- MEFs, using DNA antisense probes. For loading control, let-7a and let-7a * expression was normalized against the expression of U6 RNA.

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