The protease activity is required for 2A proto induce SGs. (A) HeLa cells were co-transfected with pmCherry-HuR and one of the plasmids (pEGFP-2AD39E, pEGFP-2AL40F, pEGFP-2AS67F, pEGFP-2AY89L, pEGFP-2AY90L, pEGFP-2AV120M, pEGFP-2AG122E, or pEGFP-2AD136N). The cells were observed at 24 h post-transfection. The percentage of EGFP-expressing cells with granules was quantified as in Figure 2A. The average of three independent experiments is indicated on the lower right of each panel. (B) HeLa cells were transfected with pEGFP-C1, or pEGFP-2AG122E. The cells were fixed at 24 h post-transfection and then stained for G3BP1. Nuclei were identified by Hoechst 33342 staining. The localization of the mCherry-HuR (A) or G3BP1 (B) proteins was determined using a fluorescence microscope (×400). (C) The protease activity of the 2Apro mutants based on the cleavage efficiency of eIF4G was determined. HeLa cells were transfected with these 2Apro mutant-expressing plasmids and the integrity of eIF4G was detected by western blotting at 24 h post-transfection.