Functional miR-snaR associates with Ago in cells. (A) Ago-IP was performed with total cell lysate extracted from HCT116 cells (WT and knockouts) transfected with pBS-snaR-A. RNAs associated with Ago were extracted and analyzed by northern blot to detect snaR-A, miR-snaR, and let-7a. Input (I) and supernatant (S) are 5% relative to the pellet (P) (nt: nucleotides). (B) Ago-IP and northern blot were performed as in A with samples from 293T, MCF-7, and MDA-MB-231 cells. Input (I) and supernatant (S) are 5% relative to the pellet (P) (nt: nucleotides). (C, top) Dual luciferase reporter for miR-snaR (pmirGLO-snaR), in which target site complementary to miR-snaR is cloned downstream from firefly luciferase, was transfected in 293T cells together with either pBS-snaR-A or pBS. (Bottom) Firefly luciferase activity was measured 48 h post transfection and normalized to Renilla luciferase activity. Error bars represent standard deviation from five experiments. All differences are statistically significant (Student's t-test, P < 0.05).
