Expression of HIS33 and deposition of H3.3 into polytene anlagen chromatin (a3) depends on the Piwi-non-coding RNA (ncRNA) pathway. The effects of Piwi knock-down on the expression of H3 variant mRNAs were studied by semiquantitative PCR (A) and quantitative PCR (B). (C) Expansion of Figure 3A: Western blot analyses of nuclear proteins isolated from micronuclei (m), macronuclei (M) and polytene anlagen (a3) as well from anlagen (a3) after Piwi-minus RNAi treatment. Antibodies used were targeted against H3.3, H3.5, H3.7, H3K4me3, H3K9ac/K14ac, H3K27me3, H3K36ac, and H3S10ph. Red arrows indicate bands corresponding to 20 kDa or 15 kDa H3 variants.