Figure 5.

Structure-Function Analysis of MEK1/MAGI1 Complexes

COS7 cells continuously growing in the presence of serum were transiently transfected with the indicated constructs.

(A) myc-tagged MAGI1 domains (schematically depicted in the upper panel) in endogenous MEK1 IPs and WCL were detected by immunoblot.

(B) myc-tagged MAGI1 and endogenous MEK1 were detected in myc-tag IPs and WCL of cells transfected with myc-tagged MAGI1 deletion mutants.

(C–E) myc-tagged MAGI1 proteins and His-tagged MEK1 were detected in myc-tag IP and WCL of cells transfected with His-tagged WT or mutant MEK1 and myc-tagged full-length MAGI1 (C), GUK (D), or WW (E) domains. Lysates were subjected to immunoblotting with His and myc antibodies.

(F and G) myc-tagged MAGI1 proteins and endogenous MEK1 were detected by immunoblotting in myc-tag IPs and in lysates of cells transfected with WT or mutant WW domains of MAGI1 (1, 2, and D, double mutant; F) or full-length MAGI1 bearing the same mutations (G). EV, empty vector.

(H and I) COS7 cells transfected with a plasmid encoding the WT or mutated GUK-WW domains of MAGI1 or EV were subjected to PTEN IP (H). The indicated antigens were detected by immunoblotting. PTEN membrane localization was assessed by immunofluorescence (I; n = 3). Values represent mean ± SD. ^∗∗p < 0.01.

The numbers in (B), (F), and (G) indicate the ratio between MEK1:myc-MAGI1 proteins, measured by densitometry. The MEK1:WT myc-MAGI1 ratio is set as 1.