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. 2011 Jun 1;25(11):1159–1172. doi: 10.1101/gad.2042311

Figure 6.

Figure 6.

Mutation of IMP2 Ser162 and Ser164 to Ala inhibits IMP2 binding to IGF2 L3 and stimulation of L3 mRNA translation. (A) Mutation of IMP2[Ser162/164] to Ala but not Asp reduces IMP2 binding to IGF2 L3 mRNA, whereas both IMP2 mutants bind mTOR similarly. Plasmids encoding Flag-tagged IMP2 wild type (WT), IMP2[Ser162Ala/Ser164Ala] (AA), or IMP2[Ser162Asp/Ser164 Asp] (DD) were transiently expressed in RD cells. The cells were extracted 24 h later, Flag-IMP2 variants were immunoprecipitated, and matched aliquots were subjected to phenol/chloroform extraction and measurement of bound IGF2 L3 mRNA by qPCR and immunoblot for the presence of endogenous mTOR. (B) Mutation of IMP2[Ser162/164] to Ala but not Asp reduces the initiation of L3-mRNA translation. RD cells stably expressing Flag-tagged IMP2 wild type (WT), IMP2[Ser162Ala/Ser164Ala] (AA), or IMP2[Ser162Asp/Ser164Asp] (DD) at levels similar to (AA) or less than (WT, DD) endogenous IMP2 (top blot) were transiently transfected with L3-firefly luciferase together with a plasmid encoding Renilla luciferase, and the translational efficiency of L3-luciferase was measured as described in the legend for Figure 2A. The effect of IMP2[Ser162Ala/Ser164Ala] on the phosphorylation of endogenous IMP2 is shown in Supplemental Figure S6.