Fig. 3
All three EhAgos associate with 27 nt sRNAs with 5′-polyP structure. a sRNA populations are bound to all three EhAgo proteins. Total RNA was prepared from anti-Myc IP using lysates from each Myc-tagged EhAgo overexpressing cell line and labeled with α-[32P]-pCp. A faint band ranging from 20 to 30 nt was noted for EhAgo2–1, a distinct 27 nt sRNA band identified for EhAgo2–2, and two sRNA populations at 27 nt and 21 nt was observed for EhAgo2–3. Arrows point to 27 nt and 21 nt sRNA bands. See also Suppl. original blots for Fig. 3a. b Western blot analysis detects a specific Myc signal for each EhAgo IP. Anti-Myc IPs along with control (anti-HA IP) were performed for three Myc-tagged EhAgo overexpressing cell lines. The Myc signal is detected at the expected size for each Myc-tagged EhAgo using anti-Myc antibody, and is absent in the control IP. The same membrane was stripped and re-probed using an anti-EhAgo2–2 antibody. The detected signal is only present in the EhAgo2–2 IP but not in the EhAgo2–1 IP nor EhAgo2–3 IP, showing the specificity of anti-Myc IP experiment. See also Suppl. original blots for Fig. 3b. c Capping assay demonstrates the 5′-polyP structure for the 27 nt sRNA populations. An increase in the sRNA size is observed for all three EhAgo sRNA populations, indicating that they have a 5′-polyP structure. The smaller sized RNAs below 24 nt size in EhAgo2–1 and EhAgo2–3 do not shift in size indicating that they do not have 5′-polyP structure. See also Suppl. original blots for Fig. 3c