983501.fig.005a
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983501.fig.005b
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Figure 5: HCS1 interacts with Arabidopsis histone H3 directly in vitro. E. coli lines that contain GST-HCS1, His-H3, or GST construct were induced to express these genes. After induction, total protein was extracted from the cell lines. Recombinant His-histone H3 protein was purified from E. coli by using metal affinity chromatography. Recombinant GST-HCS1 or GST alone was purified from E. coli by using glutathione-sepharose beads. Proteins were subjected to GST pull-down assays with GST-HCS1 or GST alone and probed by western blot to detect whether there was an interaction between HCS1 and histone H3. (a) Proteins were analyzed by 15% SDS-PAGE and stained with Coomassie Blue. I: total proteins extracted from E. coli containing a GST-HCS1 or a His-H3 construct. C: total proteins extracted from control E. coli (not containing any vector). P: recombinant GST-HCS1 or recombinant His-H3 proteins. (b) Western blot of proteins after GST pull-down to detect whether there was an interaction between HCS1 and histone H3. GST-HSC1 fusion protein and GST were detected with GST antibody; Histone H3 was detected with corresponding antibody. GST-HCS1: the proteins released from the beads coupled with GST-HCS1 after being incubated with His-histone H3 proteins; Histone H3 pulled down by GST-HCS1 was detected. GST: the proteins released from the beads coupled with GST control after being incubated with His-histone H3 proteins; no histone H3 was detected. Input His-H3: 10% of the total input recombinant His-histone H3 proteins; Histone H3 was strongly detected as expected. Experiments were conducted in triplicate.