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. Author manuscript; available in PMC: 2016 Jul 18.
Published in final edited form as: Nat Genet. 2016 Jan 18;48(3):283–291. doi: 10.1038/ng.3486

Fig. 3. Piwi binds to PRC2 in vitro but does not affect its HMTase activity.

Fig. 3

(a) Recombinant Piwi was incubated with in vitro reconstituted PRC2 complexes and subjected to immunoprecipitation by anti-IgG and anti-Piwi antibodies, followed by immunoblotting.

(b) Full-length flag-Piwi was used to pull down full-length myc-Corto, myc-Esc, myc-Su(z)12, or myc-E(z) (collectively indicated as “myc-prot”) expressed in HEK293 cell extract. Bound proteins were analyzed by immunoblotting to the myc epitope.

(c) Myc-tagged Piwi-fragment d and flag-tagged Su(z)12-fragment VI were expressed in S2 cells and used to pull down each other.

(d) Myc-tagged Piwi-fragment c and flag-tagged Esc-fragment ii are expressed in S2 cells and used to pull down each other.

(e) In vitro reconstituted PRC2 complexes (represented by E(z) immunoblotting) were incubated with or without recombinant Piwi, and 3H-methylation signals were analyzed by radiography.