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. 2014 Jan 10;9(1):e85510. doi: 10.1371/journal.pone.0085510

Figure 4. Biochemical and functional analysis of Ago2 in LRRK2 mice.

Figure 4

(A) Representative western blot analysis showing that Ago2 protein levels are not affected in the presence or absence of LRRK2 (top panel). Overexpression of human LRRK2-WT or harbouring the human PD mutation R1441G has no effect on Ago2 levels either, when compared to non-transgenic littermates (bottom panel). (B) Polysomes fractionation of LRRK2-deficient mouse brain, compared to wild-type brain. No change in Ago2 or the ribosomal protein S6 distribution was observed by the absence of LRRK2. (C) RIP-Ago2 assay of wild-type mouse brain. A representative amplification curve of miR-16 by real-time qRT-PCR shows a significant enrichment (∼500 fold) of this miRNA pulled down by RIP-Ago2. The insert demonstrates the efficiency of the immunoprecipitation (Ago2 vs. mouse IgGs). The flow-through (FT) shows the concomitant decrease of Ago2 in the post-RIP lysate. The “*” sign is Radixin, a well known non-specific protein when using the Ago2 (2A8) antibody [47]. Of note, Radixin is not immunoprecipitated by Ago2 (2A8). (D) Histogram of RIP miRNA pulled down in the presence or absence of LRRK2. In each case (n = 3), there is no difference in Ct values (qRT-PCR). LRRK2 deficiency does not affect RIP-Ago2 RNAs or miRNAs immunoprecipitation. Standard error of the mean (SEM) is shown.