Spatiotemporal occurrence of H3 variant proteins, nuclear localization of H3.7 and selected post-translational modifications (PTMs). (A) Nuclear proteins were isolated from micronuclei (m), early anlagen (a1) with visible chromatin decondensation, mid anlagen (a2) with polytene chromosomes prior to bulk DNA elimination, late anlagen (a3) at the onset of DNA elimination, DNA-poor anlagen (e) during extensive DNA elimination, and macronuclei (M). The proteins were separated by SDS-PAGE and stained with Coomassie Brilliant Blue. Red arrows indicate bands corresponding to 20 kDa (H3.7, H3.8) and 15 kDa H3 variants (H3.1 to H3.6). (B) Western blot analyses were performed using the same samples as described in (A) for SDS-PAGE. Antibodies targeted to H3.3, H3.5, or H3.7 were used for detection. (C) In situ antibody staining using primary antibodies targeted to histone H3.7 (c1 to c4) or H3K36ac (c5 to c8) (green) and DNA counterstaining (red). The cellular shape was visualized in c5 to c8 using an α-tubulin-antibody (grey). All images are confocal image stack projections of 5 to 10 images from the middle of the stacks. Abbreviations: m, micronuclei; M, macronuclei; a1 to a3, macronuclear anlagen during the first round of DNA amplification (compare above); e, macronuclear anlagen towards the DNA-poor stage; p, parental/old macronuclei. (D): Details of macronuclear anlagen (a3) using antibodies targeted to H3.7 (d1), H3K36ac (d2), H3K9ac/k14ac (d3) or H3K27me3 (d4). The lettering and color scheme is as described in (C).