Supplementary Figure 2: Sestrins as noncanonical regulator of MAPK function.

(a) Immunoblot analysis of lysates from senescent CD27^- CD28^- CD4⁺ T cells after immunoprecipitation with sestrin2 or IgG control antibodies. (b) Phospho-flow analysis of Erk, Jnk and p38 MAPK phosphorylation in cells as in Figure 1b. Pooled results (n = 4) presented relative to those of nonsenescent CD27⁺ CD28⁺ CD4⁺ T cells, set as 1. (c) Immunoblots of endogenous MKK7, MKK4 and p-MEK1/2 in cells as in (b). Total AMPK, loading control. Immunoblots representative of 3 independent experiments. (d) Validation of indicated siRNAs in primary human CD4⁺ T cells by flow-cytometry. Data (n = 2) presented relative to those cells transfected with control siRNA, set as 1. (e) MAPK signaling in nonsenescent versus senescent CD4⁺ T cells transfected with the indicated siRNAs, followed by phospho-flow analysis 48h later. Pooled data (n = 3) presented relative to those of cells transfected with control siRNA, set as 1. (f) In vitro kinase assays in sestrin2 immunoprecipitates from senescent CD4⁺ T cells transduced as indicated and studied 96h later. See also Figure 2. *p<0.05 **p<0.01 and ***p< 0.001 values were assessed by ANOVA for repeated measures with a Bonferroni post-test correction (c) or a paired Student’s t test (e-f). Errors bars depict s.e.m.