Journal: Nature immunology

Article Title: A sestrin-dependent Erk/Jnk/p38 MAPK activation complex inhibits immunity during ageing

doi: 10.1038/ni.3665

Figure Lengend Snippet: (a) Expression of surface CD27 and CD28 in human CD4 + T cells by flow-cytometry. ( b ) Immunoblots (left) and flow cytometry data (right) of endogenous sestrin1, sestrin2 and sestrin3 expression in CD4 + T cells gated as in ( a ). ( c ) 3 H-thymidine uptake and ( d ) telomerase activity in CD4 + T sen cells transduced as indicated. ( e ) DNA damage foci assessed by the DNA damage response marker γ-H2Ax in cells as above. Flow cytometry of ( f ) intracellular LCK, ( g ) CD27 and CD28 co-stimulatory receptors, ( h ) calcium abundance and ( i ) IL2 synthesis in CD4 + T sen cells transduced as indicated. Results presented relative to those of cells transduced with shCtrl, set as 1. Data are representative of four experiments ( a, b ( left )) or pooled from four experiments with four individual donors ( b ( right )) or three donors ( c , d , e , f , g , h , i ). ** p<0.01 and *** p< 0.001 calculated using ANOVA for repeated measures with Bonferroni post-test correction. Error bars indicate s.e.m.

Article Snippet: Membranes were probed with antibodies to: Sestrin1 (Genetex; Abcam), Sestrin2 (Cell Signaling), Sestrin3 (Sigma), ERK, p-ERK, p-JNK, p-p38, AMPK-α, p-AMPK-α, mTOR S6K1, MKK7, MKK4, p-MEK1/2 and GAPDH (all from Cell Signaling).

Techniques: Expressing, Flow Cytometry, Western Blot, Activity Assay, Marker, Transduction

Journal: Nature immunology

Article Title: A sestrin-dependent Erk/Jnk/p38 MAPK activation complex inhibits immunity during ageing

doi: 10.1038/ni.3665

Figure Lengend Snippet: (a) Immunoblot analysis of lysates from CD4 + T sen precipitated with IgG control or sestrin1 antibodies, and stained for the indicated proteins. Right margin, molecular weights. ( b ) MAPK phosphorylation of AMPK immunoprecipitates from CD4 + T sen transduced as indicated then treated with the AMPK agonist A-769662 (150 μM, 60’) 96h later. Results assessed by in vitro kinase assay based on an enzyme-linked immunosorbent assay (absorbance at 450 nm) and presented relative to that of cells transduced with shCtrl, set as 1. ( c ) MAPK auto-phosphorylation in sestrin1 complexes from CD4 + T sen incubated for 30’ with the AMPK agonist A-769662 (150 μM, 30’), the Erk inhibitor FR18024 (20 μM), the Jnk inhibitor SP-600125 (10 μM) or the p38 inhibitor SB-203580 (10 μM), in the presence or absence of ATP (200 μM). MAPK activity was determined and presented as in ( b ). ( d ) ELISA-based ATP abundance in AMPK-γ immunoprecipitates from CD4 + T sen whereby one or more sestrins were silenced by shRNA. Results presented relative to that of cells transduced with shCtrl, set as 1. ( e ) AMPK phosphorylation of cells as in ( d ); GAPDH, loading control. ( f ) Elisa-based MAPK-binding assays in sestrin1 immunoprecipitates from cells as in ( a ) transfected with either Ctrl or siAMPK-γ for 36h, and presented relative to those of cells transfected with siCtrl, set as 1. Data are representative of two ( a ) or three ( e ) experiments or pooled from three independent experiments with three individual donors ( b , c , d, f ). * p<0.05 ** p<0.01 and *** p< 0.001, ANOVA for repeated measures with Bonferroni post-test correction. Error bars indicate s.e.m. throughout.

Article Snippet: Membranes were probed with antibodies to: Sestrin1 (Genetex; Abcam), Sestrin2 (Cell Signaling), Sestrin3 (Sigma), ERK, p-ERK, p-JNK, p-p38, AMPK-α, p-AMPK-α, mTOR S6K1, MKK7, MKK4, p-MEK1/2 and GAPDH (all from Cell Signaling).

Techniques: Western Blot, Control, Staining, Phospho-proteomics, In Vitro, Kinase Assay, Enzyme-linked Immunosorbent Assay, Transduction, Incubation, Activity Assay, shRNA, Binding Assay, Transfection

Journal: Nature immunology

Article Title: A sestrin-dependent Erk/Jnk/p38 MAPK activation complex inhibits immunity during ageing

doi: 10.1038/ni.3665

Figure Lengend Snippet: ( a ) Immunoblot analysis and ( b ) pooled data ( n = 4 ) of Erk, Jnk, and p38 phosphorylation in lysates from CD4 + T cells from 20-month old vaccinated Sesn1 +/- and Sesn1 -/- mice following immunoprecipitation with sestrin2 antibodies. Results in ( b ) presented relative to those of Sesn1 +/- mice, set as 1. Sestrin2 expression in whole cell lysates (WCL), loading control. Sestrin1 expression is also shown. ( c ) Image-Stream analysis and ( d ) % co-localization scores of sestrin2 + -pMAPKs + in CD4 + T cells from Sesn1 -/- and Sesn1 -/+ mice, five days post-vaccination with FLUAD, presented as in ( b ). In vitro MAPK assays of sestrin2 immunoprecipitates from mouse Sesn1 -/- CD4 + T cells incubated for 60’ with or without ( e ) recombinant human sestrin1 or ( f ) the AMPK agonist A-769662, in the presence or absence of ATP. Results presented relative to that of non-reconstituted Sesn1 -/- cells (‘basal’), set as 1. In ( e ) cells were transfected with the indicated siRNAs 48h before lysis. Data are representative of four experiments ( a, c ) or pooled from four experiments with four (age-matched) individual mice ( b , d , e , f ). ** p<0.01 and *** p< 0.001, ANOVA for repeated measures with Bonferroni post-test correction. Error bars indicate s.e.m. throughout.

Article Snippet: Membranes were probed with antibodies to: Sestrin1 (Genetex; Abcam), Sestrin2 (Cell Signaling), Sestrin3 (Sigma), ERK, p-ERK, p-JNK, p-p38, AMPK-α, p-AMPK-α, mTOR S6K1, MKK7, MKK4, p-MEK1/2 and GAPDH (all from Cell Signaling).

Techniques: Western Blot, Phospho-proteomics, Immunoprecipitation, Expressing, Control, In Vitro, Incubation, Recombinant, Transfection, Lysis