Skip to main content
. 2003 Dec;23(24):9117–9126. doi: 10.1128/MCB.23.24.9117-9126.2003

FIG. 2.

FIG. 2.

Analysis of mutations at d4E-BP phosphorylation sites. (A) Mutation of Thr37 and/or Thr46 to Ala (T37A and T46A, respectively) prevents the insulin-induced phosphorylation of d4E-BP. As expected, the double mutant (T37A/T46A) also showed no phosphorylation. The last lane (-) is an untransfected control. WT, wild type; +, treated; −, untreated; P, phosphorylated. (B) Mutation of Ser65 and/or Thr70 to Ala (S65A and T70A, respectively) had no effect on phosphorylation. As expected, the double mutant (S65A/T70A) also had no effect. S2 cells transfected with 3HA-tagged d4E-BP mutant cDNAs were starved for 24 h before insulin treatment. The effect of insulin was assessed with anti-phospho-4E-BP1(Thr37/46) (α4E-BP1 P-T37/46) (middle), and protein expression levels were compared using anti-HA-11 (αHA) (bottom). Total protein loading levels were compared using anti-deIF4A (αdeIF4A).