Fig. 4

Hsp90 and Mas5 are required for the formation of siRNA-containing Ago1 complexes. a Detection of proteins in whole-cell extracts by western blotting. Antibodies against FLAG epitope, Hsp90, and α-tubulin were used. b Detection of pericentromeric siRNA. Equal amounts of total RNA extracted from untagged cells or from cells expressing FLAG-Ago1 were loaded into each lane. Pericentromeric siRNA (cen siRNA) was detected by northern blotting with specific probes (see “Methods” section). U6 snRNA was used as a loading control. c Detection of pericentromeric siRNA in Ago1 complex. FLAG-Ago1 complex was immunoprecipitated with an antibody against the FLAG epitope. Pericentromeric siRNA extracted from the immunoprecipitates was detected by northern blotting (cen siRNA). Signal intensities of the pericentromeric siRNA relative to the wild-type FLAG-Ago1 sample are indicated. Successful immunoprecipitation was validated by means of western blotting with an antibody against the FLAG epitope (FLAG-Ago1). Untagged cells were used as negative controls. d, e Detection of FLAG-Ago1 in Arb1-Myc (d) and Tas3-Myc (e) immunoprecipitates. Soluble extracts (Input) and immunoprecipitates (Myc-IP) were separated on SDS-PAGE and detected with antibodies against the Myc and FLAG epitopes; the loading control protein was detected with an antibody against α-tubulin. Asterisks, background signals. Signal intensities of FLAG-Ago1 normalized with those of Arb1-Myc are indicated