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. 2011 Dec 15;7(12):e1002447. doi: 10.1371/journal.ppat.1002447

Figure 3. GrB inhibits in vitro translation of Luc.

Figure 3

IVTT was used to synthesize Luc in the presence of MetS35. Labeled Luc protein was visualized by autoradiography. (A) A range of GrB concentrations (0–30 nM) were added to the IVTT master mix to test their effect on transcription/translation; (n = 3 of 3 independent experiments). (B) Detectable Luc was compared in the absence of GrB, and in the presence of 30 nM GrB added before or after IVTT; (n = 3 of 3 independent experiments). (C) Purified Luc mRNA (10 µg/ml) was used to synthesize MetS35-labeled Luc. 30 nM GrB prevents translation; (n = 3 of 3 independent experiments). (D) Two different concentrations of proteases (3 and 30 nM) GrB, granzyme A (GrA), granzyme K (GrK), Caspase-3 (Casp-3), Caspase-7 (Casp-7) and Caspase-8 (Casp-8) were tested in IVTT of Luc; (n = 3 of 3 independent experiments). (E) Luminescence generated by Luc in IVT reactions treated with 30 nM proteases, where sham-treated IVT reactions were considered 100%; Statistical significance: p<0.01 (**) and p<0.005 (***); (n = 3 of 3 independent experiments).