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Figure S1. H3 and H3.3 Regulation during Early Development, Related to Figure 1
(A) H3 and H3.3 expression levels by Northern Blot. We extracted total RNAs from embryos at indicated developmental stages for analysis of H3.2 and H3.3 transcripts by Northern Blot. We show the signals for total H3 transcripts (H3.2+H3.3 cDNA), total RNA visualized by methylene blue (revealing the 28S and 18 s rRNAs), H3.2 and H3.3 single transcripts (H3.2b+H3.3b 3′UTR) and single H3.3 transcript (H3.3b 3′UTR).
(B) Expression pattern of H3.2 and H3.3 mRNA during Xenopus development. We fixed embryos at indicated stages and performed whole mount in situ hybridization using DIG-UTP RNA probes. We show the expression pattern of total H3 (H3.2+H3.3 cDNA), H3.2a, H3.3b and HIRA single transcripts (3′ UTR). The Sox17α probe served as a reference for endoderm staining, and a sense probe was used as a negative control. We sectioned stage 9 and 11 embryos prior to the in situ hybridization procedure. Scale bars: 1 mm, sketches adapted from (Nieuwkoop and Faber, 1967).
(C) Soluble pool of H3.2 versus H3.3 levels throughout development. We prepared HSE extracts from eggs and embryos at indicated stages for analysis by Triton Acid Urea (TAU) gel electrophoresis. This allowed the separation of H3.2 and H3.3 variants that we revealed by Western blot with an H3 antibody. Memcode staining served as a loading control, MBT = Mid Blastula Transition, sketches adapted from (Nieuwkoop and Faber, 1967).
