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. 2015 Jan 14;34(4):559–577. doi: 10.15252/embj.201490062

Figure 4.

Figure 4

Coi12p is necessary for the loading of scnRNAs to Twi1p in cell lysates
  1. A schematic drawing of the scnRNA loading assays in cell lysates. The circled samples (B-i, B-ii, B-iii, and B-iv) correspond to the lanes shown in (B). The boxed samples (C-i, C-ii) correspond to the lanes shown in (C). See text for details.
  2. The 27-nt RNAs that co-precipitated with Twi1p from lysates with the different conditions shown in (A) were separated on a denaturing gel and detected by autoradiography (top). Precipitated Twi1p was detected by Western blot using an anti-Twi1p antibody (bottom).
  3. Proteins in the cell lysate before (i) and after (ii) immunodepletion of Coi12p were analyzed by Western blot using anti-Coi12p (top) and anti-Twi1p (bottom) antibodies.
  4. Cell lysate from the cells expressing FLAG-HA-tagged wild-type Twi1p (WT) or Twi1p in which the aspartic acid 526 was replaced with asparagine (D526N) at 3 hpm was incubated with radiolabeled double-stranded 27-nt RNAs. The Twi1p-containing complex was immunoprecipitated with an anti-Twi1p antibody, co-precipitated RNAs were separated on a native gel, and 27-nt RNAs were detected by autoradiography. As markers, ssRNA1 and ssRNA2, which were used for producing the radiolabeled double-stranded 27-nt RNAs, were mixed in 2:1 (left) or 1:2 (right) ratio, allowed to form double-stranded RNAs, and were separated in the same native gel.