Figure 7
Cellular localization and association of miR-136 with RIG-I.
(A) A549 cells on coverslips were transfected with FAM-labeled miR-136 (30 nM) and Flag-RIG-I plasmids (1 μg). 24 hours after transfection, cells were formaldehyde fixed and analysed by Immunofluorescence assay using antibody against Flag. Colocalization was analysed using Image J. (B) 293T cells in 75 cm2 flask were transfected with Flag-RIG-I plasmids (20 μg) for 36 hours. Total cellular protein was incubated with biotin-labeled miR-136 or negative control (8 μg) and subjected to streptavidin beads binding. Bound RIG-I protein was analysed by western blot. (C) RIP experiments were performed as described in methods section. The immunoprecipitated RNAs were subjected to RT-PCR assay for CT mean calculation and gel analysis. (D) The nucleotide sequences of RT-PCR product of miR-136 were verified by sequencing analysis. UD, Undetermined; B-miR136, biotin-miR136; B-NC, biotin-negative control. The blots/gels were run under the same experimental conditions. Full-length blots/gels are presented in Supplementary Figures 9, 10.
