Inhibition of PP2A_B56δ by O₂⁻ is mediated by the nitration of B56δ at Y289. (A) Nitration status of B56δ^Y289 (denoted as 3NO₂-Y289) was evaluated in Jurkat cells subjected to the indicated treatments using a custom-made Ab raised in rabbits immunized with peptides containing nitrated B56δ^Y289. The detected protein band migrates at a similar rate as endogenous B56δ on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Nitration of B56δ did not appear to alter the migration rate of B56δ because only a single band was detected by B56δ antibody. (B) Jurkat cells expressing WT-B56δ or Y289F-B56δ were treated with DDC for 4 or 8 hours and then lysates were IB for the indicated proteins. pCEP4, empty vector. (C) Jurkat cells expressing WT-B56δ or Y289F-B56δ were treated with 8 hours DDC, lysed, IP using anti-HA Ab, and IB for the indicated proteins. (D) WT-B56δ– or Y289F-B56δ–expressing Jurkat cells were pretreated for 1 hour with DDC and then treated with either 2.5 μM Eto or 1 μM Doxo for 18 hours. Cell viability was assessed via MTT assay thereafter. (E) Jurkat cells were transfected with first with SOD1-targeted siRNA (siA or siB), and 24 hours later, with plasmids expressing WT-B56δ or Y289F-B56δ. Twenty-four hours later, lysates were subjected to co-IP analysis as in panel C. (F) Jurkat cells were subjected to the same transfection procedures as in panel E, followed by 18 hours of Eto (2.5 μM) or Doxo (1 μM) treatment. Cell viability was then assessed via MTT assay.