Figure 1

Enzymatic cleavage of H3 interferes with the measurement of certain histone modifications. (A) Known enzymatic cleavage sites in H3 for mouse ESCs [11]. Bold solid lines indicate sites that are frequently cleaved, thin solid lines indicate sites that are less frequently cleaved, and dotted lines indicate sites that are rarely cleaved [11]. (B) Western blot (Odyssey® CLx Infrared Imaging System, Li-Cor) was used to measure total H3 protein levels (Sigma, H0164, 1:4,000) in 11 representative histone samples that had been isolated, using an acid-extraction method [27], from PBMCs collected from arsenic-exposed Bangladeshi adults enrolled in the Folic Acid and Creatine Trial (FACT), a randomized controlled trial of folic acid and creatine supplementation; sample collection and processing for this study has been described previously [28]. The expected size of H3 is ~17 kDa. A distinct cleavage product of H3 is observed at ~15 kDa, and an additional H3 cleavage product between 15 and 17 kDa is also present in several of the samples (top panel). In the same 11 samples, three histone modifications that are located in different regions of H3 were assessed by Western blot: H3K9me2 (Abcam, ab1220, 1:1,000, mouse) (second panel), H3K36me2 (Abcam, ab9049, 1:1,000, rabbit) (third panel), and H3K79me2 (Abcam, ab3594, 1:400, rabbit) (fourth panel).