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. 2011 Feb 22;6(2):e17362. doi: 10.1371/journal.pone.0017362

Figure 1. Hydroxyurea and nocodazole affect c-Myb levels and activity.

Figure 1

(A) Conventional ChIP assay with Jurkat cells. Asynchronously growing Jurkat cells were fixed and subjected to ChIP analysis to determine whether c-Myb was associated with the CCNB1 and CCNE1 promoters. The results are plotted as fold enrichment compared to control IgG as measured using Quantitative real-time PCR (QPCR). Error bars show the standard deviation of triplicate QPCR assays. The results are representative of several independent experiments. (B) Effects of hydroxyurea (HU) and nocodazole (NOC). Jurkat cells were treated 16–18 hr with the cell cycle blockers HU or NOC to arrest the cells in S phase or G2/M, respectively, then samples were stained with Hoechst 33342 and the cell cycle profiles were compared to untreated, asynchronously (Asyn) growing cells by flow cytometry. The histograms show cell number vs DNA content, and the G1, S and G2/M portions of the diagrams are labeled. (C) ChIP assay with HU and NOC treated cells. Jurkat cells treated 16–18 hr with HU or NOC were harvested, fixed and processed for ChIP assays, using anti-Myb antibodies. The association of c-Myb with the CCNB1 or CCNE1 promoters was assessed by QPCR, as described above. Results are plotted as fold enrichment compared to control IgG and error bars show standard deviation of triplicate QPCR assays. These results are representative of several independent experiments. (D) Expression of c-Myb protein. Total cell extracts from Jurkat cells growing asynchronously (Asyn) or treated with NOC or HU were analyzed by Western blot for expression of c-Myb protein. The blot was treated with one anti-Myb antibody, then stripped and re-probed with a different anti-c-Myb antibody and finally stripped and re-probed for beta-actin as a protein loading control. These results are representative of several independent experiments. Note: All of the experiments in this figure were performed at least three times each. The results shown are from a single experiment but are representative of all the trials.