ONOO⁻ is downstream of O₂⁻ in the inhibition of PP2A_B56δ holoenzyme assembly. (A) Intracellular NO was measured in DDC-treated Jurkat cells using a NO-sensitive probe, DAF-FM. (B) Jurkat cells were pretreated with FeTPPS for 1 hour, followed by DDC treatment, and then IB for the indicated proteins. (C) S70pBcl-2 status was assessed in Jurkat cells after a 2-hour treatment with the indicated concentrations of ONOO⁻. (D) Jurkat cells were treated with 10 μM ONOO⁻ for 2 hours and harvested for co-IP assay with Bcl-2 as bait. (E) IB analysis of mitochondrial fractions from Jurkat cells preincubated with 100 μM FeTPPS followed by DDC or ONOO⁻ treatment. (F) Multiple sequence alignment of B56 regulatory subunits of both mammalian and nonmammalian origin. The nitration-prone tyrosine residue (Y) identified in B56 family members is boxed in red. Blue arrows denote residues that are implicated in the interaction of B56 subunits with PP2A-A.²⁸  Adjacent residues proposed to predispose a tyrosine residue toward nitrative modification process are indicated by red, green, and purple arrows (refer to supplemental Figure 5C for details). Multisequence alignment analysis was performed via Jalview 2.7. (G-H) Lysates from DDC (with or without FeTPPS pretreatment) and ONOO⁻-treated Jurkat cells were IP using anti-B56δ Ab and IB for 3-NT and other indicated proteins. (I) Twenty-four hours after SOD1 silencing, Jurkat cells were treated with 2.5 μM DPI for an additional 24 hours and harvested for co-IP analysis using the indicated Abs. (J) Lysates from bSOD1 treated (16 hours, 1 kU) or SOD1-overexpressing Jurkat cells were IP using anti-B56δ Ab and IB for 3-NT and other indicated proteins.