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. 2002 Apr 15;157(2):219–229. doi: 10.1083/jcb.200110045

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Copyright © 2002, The Rockefeller University Press

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Figure 6. — AIR-2 phosphorylates REC-8 in vitro. (A) Kinase assays were performed with GST–AIR-2 (AIR-2; lanes 1, 3, and 5) or kinase-dead mutant GST–AIR-2 (AIR-2D; lanes 2, 4, and 6) using GST–REC-8 (lanes 1 and 2), GST–COH-1 (lanes 3 and 4), or GST–COH-2 (lanes 5 and 6) as substrates. Two bands were pulled down with glutathione beads in lanes 1 and 2. The slower migrating band is the full-length REC-8, whereas the faster migrating band is truncated REC-8. AIR-2,* GST–AIR-2 autophosphorylation; AIR-2_P, Ponceau staining of AIR-2 protein; substrate,* phosphorylation of corresponding test substrates; substrate_P, Ponceau staining of each substrate protein. (B) Kinase assays with either wild-type (lanes 1 and 5), T625A (lanes 2 and 6), S626A (lanes 3 and 7), or T625A/S626A (lanes 4 and 8) REC-8 using wild-type AIR-2 (lanes 1–4) or kinase-dead AIR-2 (lanes 5–8). The top half is phosphorimaging and the bottom half is Ponceau staining. Full-length REC-8, AIR-2, and breakdown products are as indicated to the left.