Figure 6

MEF2C-S396A is less efficiently sumoylated in vitro and in vivo. (A) The recombinant GST-MEF2C-ΔN2-WT or GST-MEF2C-ΔN2-S396A proteins were subjected to CDK1 kinase assays and analyzed by SDS-PAGE followed by autoradiography. (B) 0.75 μg of GST-MEF2C-ΔN2-WT, S396A, or S396E proteins were incubated with 1 μg of E1, 0.5 μg of E2, and 1.25 μg of His₆-SUMO1 for the indicated time and stopped by SDS-PAGE sample buffer. The samples were resolved by SDS-PAGE and blotted with anti-MEF2C. (C) 0.3 μg of GST-MEF2C-ΔN2-WT, S396A, or S396E proteins were subjected to CDK1 kinase assays and then incubated with 0.2 μg of E1, 0.1 μg of E2, and 0.3 μg of His₆-SUMO1 for 15 min and stopped by SDS-PAGE sample buffer. The samples were resolved by SDS-PAGE and blotted with anti-MEF2C. (D) 10T1/2 cells were transfected with the indicated plasmids. Cell lysates were resolved by SDS-PAGE and blotted with anti-MEF2C. (E) Sequence alignment of the sumoylation sites of several SUMO substrates. Conserved amino acids were shown in bold.