Fig. 9. Activation of LKB1 by association with STRAD and MO25 isoforms. 293 cells were transfected with constructs encoding GST–LKB1 in the presence or absence of constructs encoding the indicated isoforms of STRAD and MO25. Thirty-six hours post-transfection, GST–LKB1 was affinity purified and assayed for autophosphorylation and transphosphorylation of MBP as described in the Materials and methods. The reactions were electrophoresed and the gel autoradiographed (top panel). Phosphorylation of MBP by LKB1 at Thr65 was also monitored by immunoblotting with a phosphospecific antibody (T65-P), which recognizes MBP phosphorylated by LKB1 at this residue. Each reaction was also immunoblotted with the appropriate epitope tag antibodies to monitor levels of LKB1, STRAD and MO25 isoforms. Similar results were obtained in three separate experiments.