Fig 4.
Role of ankyrin repeats on LegAS4 activity. (A) Comparison of the methyltransferase activity of the SET domain or full-length LegAS4 against histone H3 at Lys14. In vitro HMT assay of 500 ng of GST-tagged enzymes in the presence of 500 ng of histone H3. The reaction was performed for 30 min at 30°C in the presence of cold SAM. GST-tagged proteins were detected with anti-GST antibodies, methylation of histone H3 by using specific H3K14me antibodies and H3 detected in the ponceau staining. (B) In vitro binding of the SET domain or full-length LegAS4 to H31-21aa (P1) ,H31-21aaThr6Tyr (P2), and H31-7aa (P3). I, input; p0, no peptide. Biotin-conjugated histone peptides were immobilized on Streptavidin-coated beads and their binding to GST-tagged proteins detected by using specific anti-GST antibodies. (C) Comparison of methyltransferase activity of LegAS4 or LegAS4-Asn493Tyr against histone H3 at Lys14. In vitro HMT assay of 500 ng of GST-tagged enzymes in the presence of 500 ng of histone H3. The reaction was performed for 30 min at 30°C in the presence of cold SAM. GST-tagged proteins were detected with anti-GST antibodies, methylation of histone H3 by using specific H3K14me antibodies, and H3 detected in the ponceau staining. (D) In vitro binding of LegAS4 or LegAS4-Asn493Tyr to H31-21aa (P1), H31-21aaThr6Tyr (P2), and H31-7aa (P3). I, input; p0, no peptide. Biotin-conjugated histone peptides were immobilized on Streptavidin-coated beads and their binding to GST-tagged proteins detected by using specific anti-GST antibodies.