Figure 2. GrB-mediated cleave of eIF4G3 occurs at position D¹⁴⁰⁸.

(A) Autoradiography of eIF4G3 treated with 30 nM GrB for 30 min. Treatment of eIF4G3 (∼200 kDa) with 30 nM GrB generates a proteolytic fragment; (n = 3 of 3 independent experiments). (B) The eIF4G3 isoform, eIF4G1, is not degraded by GrB. Loaded protein levels for eIF4G1 and eIF4G3 lanes are identical to their corresponding GrB treatment lanes; (n = 3 of 3 independent experiments). To visualize eIF4G3 fragments, eIF4G3 lanes were loaded at a higher concentration. (C) Putative P1 residue D¹⁴⁰⁸ of eIF4G3 was mutagenized to alanine to generate the mutant eIF4G3^Δ. eIF4G3 and eIF4G3^Δ were synthesized in vitro and labeled with Met^S35. These two proteins were treated with 30 and 300 nM GrB. Detectable degradation products are indicated with an arrow; (n = 3 of 3 independent experiments).