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  • br concentrations of FeTPPS a ONOO decomposition catalyst In


    concentrations of FeTPPS, a ONOO− decomposition catalyst. Indeed, a dose-dependent decrease in S70pBcl-2 was observed (Fig. 4b), thereby prompting us to explore the involvement of PP2A. To that end, results show that B56δ subunit recruitment to the catalytic subunit (C-subunit) of PP2A was inhibited in M14 Rac1V12 Epirubicin HCl (Fig. 4c). IgG control is displayed in Supplementary Fig. S6. Furthermore, we provide evidence that, similar to our recent findings in hematopoietic cells [5], B56δ is also the regulatory subunit that binds to S70pBcl-2 (for its subsequent dephosphorylation) in M14 cells (Fig. 4d). Supporting that, a significant increase in S70pBcl-2 is detected in cells upon B56δ knockdown (Fig. 4d; Input). To further corroborate our co-IP data on B56δ/C-sub-unit interaction, PLA with B56δ and C-subunit proteins in M14 pIRES cells showed significantly more interacting signals (red) as compared to that of Rac1V12 (Fig. 4e–f). Importantly, treatment of ABT199, shown to prevent the binding of Bcl-2 to Rac1 (Fig. 2f), enhanced the binding of B56δ to its C-subunit, while concurrently interacting with Bcl-2 in CEM/Bcl-2 cells (Fig. 4g). Similarly, tiron could prevent the inhibition of B56δ recruitment to the C-subunit, while in the presence of Bcl-2, in M14 Rac1V12 cells (Fig. 4h). Collectively, these data implicate Rac1/ Bcl-2 interaction in the redox-mediated inhibition of PP2A assembly and S70pBcl-2 dephosphorylation.
    Intriguingly, scavenging Rac1-induced O2.- not only reduced S70pBcl-2 but also decreased Rac1/Bcl-2 interaction (Fig. 5a), thereby suggesting that S70pBcl-2 could have an additional role in stabilizing the interaction between the two proteins. To verify this, we transiently overexpressed wild-type (WT) Bcl-2 in M14 cells and artificially clamped down S70pBcl-2 via pharmacological inhibition of JNK (SP600125); JNK phosphorylates Bcl-2 independent of O2.-. A dose- 
    dependent decrease in S70pBcl-2 and downstream target of JNK, phospho-c-Jun, were observed following 6hour-treatment of SP600125 (Fig. 5b, S7a). Similar effects of JNK inhibition were observed in M14 Rac1V12 cells (Fig. S7b). Importantly, the decrease in S70pBcl-2 also correlates with the decrease in Rac1/Bcl-2 interaction (Fig. 5c), thereby suggesting that S70pBcl-2 could further stabilize this protein-protein interaction.
    To ascertain this hypothesis, we performed co-IP-Western analyses upon transient overexpression of WT Bcl-2, phosphorylation-inefficient mutant (S70A) or phosphomimetic mutant (S70E) in M14 cells. Our data show a significant increase in S70pBcl-2 in S70E-transfected cells, and most noticeably strong Rac1/Bcl-2 interaction (Fig. 5d), which was absent in S70A-transfected cells (Fig. 5e). It is also worth mentioning that the increase in Rac1/Bcl-2 interaction in WT Bcl-2-transfected cells could be attributed to the increased endogenous S70pBcl-2 (Fig. 5d–e; inputs). Notably, as S70E mutant displayed an enhanced S70pBcl-2 compared to WT Bcl-2, this suggests that the S70 phospho-specific antibody could cross-react with S70E phosphomimetic mutant. This is supported by the authenticity of the antibody as Bcl-2 knockdown could eliminate S70pBcl-2 in Jurkat cells expressing WT Bcl-2 or S70E as well as sequencing of S70E plasmid verified the site-directed mutation (Figs. S8a–b).
    Corroborating our co-IP data, PLA with Bcl-2 and Rac1 proteins in CEM cells transiently overexpressing the S70E and WT Bcl-2 showed significantly more interacting signals (red) as compared to that of S70A and pcDNA3.1 (Fig. 5f–g, S8c; successful transfection). Collectively, these findings indicate an additional role of S70pBcl-2 in securing the interaction between Rac1 and Bcl-2, thereby suggesting a positive feedforward loop that involves the interaction between active Rac1 and Bcl-2 to permit redox-mediated inactivation of PP2A and subsequent accumulation of S70pBcl-2, which in turn further secures the