Figure 7: Inactivation of GSK3β by p38 MAPK promotes accumulation of the prosurvival factor Mcl-1.

(a,b) WT and GSK3β-KI B cells were activated for two days and the expression of β-catenin (a), Mcl-1, P-S³⁸⁹ GSK3β and total GSK3β (b) were examined by western blotting. GAPDH is shown as a loading control. (c) WT and GSK3β-KI B cells were activated as in a and Bclx, Bcl2, Bid, Bax and Bad levels were examined by western blotting. (d) Western blot analysis for PUMA in irradiated WT thymocytes (5 Gy) and activated WT and GSK3β-KI B cells. (e,f) B cells activated as in a were stained with MitoTracker (e) or TMRE (f) and analysed by flow cytometry. The number represents the percentage of cells within the gate. (g) WT and GSK3β-KI B cells were examined by western blotting for the expression of cleaved caspase-3 (activated caspase-3), full length RIPK1 (RIPK1) and cleaved RIPK1 (cleaved RIPK1). (h) WT and GSK3β-KI B cells were activated, after 2 days Nectrostatin-1s (Nec-1s) was added and cell viability was determined by cell counting 24 h later (n=3). (i) WT and GSK3β-KI B cells were activated for 3 days and phospho-MLKL was examined by western blotting. (j) WT and GSK3β-KI B cells in the presence or absence of the GSK3 inhibitor (GSK3-Inh) were activated and examined by immunostaining and confocal microscopy for Mcl-1 (red) and TOPRO nuclear stain (blue). Scale bar, 3 μm. (k) Mcl-1 levels in nuclear (Nuc) and mitochondrial (Mito) extracts from activated WT and GSK3β-KI B cells were determined by western blot analysis. Histone and CoxIV (Complex IV) were used as loading controls. (l) WT and GSK3β-KI B cells were transduced with either an empty retrovirus (E), a retrovirus expressing wildtype Mcl-1 (Mcl) or a retrovirus expressing a Ser¹⁴⁰Ala mutant of Mcl-1 (mMcl). Three days after activation cell viability was determined by cell counting. (n=3) and *P value<0.05 as determined by t- test (h) or one-way ANOVA (l). Data are representative of three or more independent experiments.