Supplementary Figure 2: Ago1 D580A, Ago1 F276A R773E, Ago1 F276A and Ago1 R773E are null-mutant proteins whose small RNA–binding activity correlates with their assembly into RITS.

(a) Schematic illustrating the ectopic insertion of 3xFLAG-tagged alleles of ago1 with endogenous promoter and terminator sequences near the trp1⁺ locus on chromosome 2. The native ago1⁺ locus harbors either a wild-type untagged allele or a deletion. (b) Tenfold serial dilutions of otr1R::ura4⁺ pericentromeric silencing reporter cells of the indicated genotypes, plated on non-selective medium or medium containing 5-FOA. (c) Northern blot analysis of dg siRNAs in total small RNA fractions and RNA immunoprecipitated with wild-type and mutant 3xFLAG-Ago1 proteins, in cells also expressing untagged wild-type Ago1, and Western blot analysis of input extracts and FLAG-immunoprecipitated material. (d) Western blot analysis of co-immunoprecipitation experiment to assay association of Tas3 with the indicated Ago1 proteins. (e) Western blot analysis of co-immunoprecipitation experiment to assay association of Arb1 with the indicated Ago1 proteins.