FIG. 3.

Cytotoxicity assay in yeast strain INVSc1 transformed with the plasmid pYES-522HS (codon-optimized RT0522), pYES-S86A (site-directed mutagenesis of codon-optimized RT0522 at Ser 86 to Ala), or pYES-S86A-D250A (site-directed mutagenesis of codon-optimized RT0522 at Ser 86 to Ala and Asp 250 to Ala). (A) Western blot analysis of the expression of codon-optimized RT0522 and its mutant derivatives in yeast strain INVSc1 under inducing conditions (SC-U+Gal medium). The total proteins from yeast cells carrying the appropriate plasmid were probed with anti-V5 antibody (Invitrogen) using the Western Breeze chemiluminescent immunodetection kit (Invitrogen). Lane 1, MagicMark XP Western protein standard (Invitrogen); lane 2, pYES-522HS/INVSc1; lane 3, pYES-S86A/INVSc1; lane 4, pYES-S86A-D250A/INVSc1; lane 5, pYES2/CT/INVSc1 (vector control). The sizes of the expected protein (including C-terminal V5 epitope and 6×His tag) RT0522 (lane 2) and its mutant derivatives (lanes 3 and 4) are shown on the right (69.5 kDa). (B) Transformed yeast cells were streaked onto inducing (SC-U+Gal) or repressing (SC-U+Glu) agar and incubated at 30°C for 3 days. (C) Transformed yeast cells with the plasmids pYES-522HS or pYES/CT/LacZ (control plasmid) were streaked onto inducing (SC-U+Gal) agar containing PLA₂ inhibitor MAPF at 15 μM or BEL at 20 μM and incubated at 30°C for 3 days.