Figure 4. GrB resistant eIF4G3^Δ can restore in vitro translation of Luc.

(A) Translation of Luc mRNA was monitored by autoradiography. In the presence of 30 nM GrB, translation of Luc is blocked. Purified eIF4G3^Δ -HA (0.01–2 µg/ml) was added the reaction after GrB treatment to recover Luc in vitro synthesis. Luc shows as a band at 61 kDa and radio-labeled eIF4G3^Δ -HA shows as a band at ∼200 kDa; (n = 3 of 3 independent experiments). (B) Same experiments as in (A) but with purified wild-type eIF4G3-HA; (n = 3 of 3 independent experiments). (C) Luminescence generated by Luc IVT in (A) was measured and expressed as %Luc luminescence, where expression of Luc with no treatment was considered 100%. Densitometric analysis was conducted for eIF4G3^Δ -HA, and expressed as %eIF4G3^Δ -HA densitometry, where densitometric values for Luc with no treatment was considered 100%; (n = 3 of 3 independent experiments). (D) GrB activity colorimetric assay. GrB inh and purified serpina 3n were used as positive controls to inhibit GrB activity. Three different concentrations of eIF4G3^Δ (0.1, 1 and 2 µg/ml) did not inhibit GrB activity; (n = 3 of 3 independent experiments).