Figure 4: Survival of thymocytes undergoing TCRβ rearrangement at the DN3 stage of development is dependent of inactivation GSK3β by phosphorylation on Ser³⁸⁹.

(a) Diagram depicting the GSK3β Ser³⁸⁹Ala knockin (KI) construct, the endogenous GSK3β allele containing exon 11 (black box), the targeted allele with the neomycin selection cassette still present and the targeted allele with the neomycin cassette removed by Cre recombinase. White notched arrow heads represent LoxP sites. (b) Whole-cell lysates from thymocytes of WT, heterozygous (Het) and homozygous GSK3β-KI mice were analysed by western blot analysis for P-Ser³⁸⁹ GSK3β, P-Ser⁹ GSK3β and total GSK3β. (c) GSK3β activity in DN thymocytes and mature CD4 splenocyte from WT and GSK3β-KI mice were examined by in vitro kinase assays (n=3). (d) Total number of thymocytes in WT (circles) and GSK3β-KI mice (squares) (n=8). (e) Total numbers of DN3 and DN4 thymocytes and the ratio of DN3/DN4 thymocytes from WT and GSK3β-KI mice (n=6). (f) Intracellular staining of TCRβ and flow cytometry analysis of DN3 thymocytes from WT and GSK3β-KI mice (n=4). The percentage of iTCRβ positive cells within the DN3 population is shown. (g) The percent fluorescence signal intensity of KI Vβ CDR3 region peaks relative to WT Vβ CDR3 region peaks (n=3 independent mice). (h) Real-time PCR was used to examine recombination between Vβ (TRBV19 and TRVB16) and Jβ (Jβ1.1 or Jβ2.1) in genomic DNA from DN thymocytes. Signals were normalized to the non-recombining gene. (n=3 independent mice for each genotype). (c,e,f,h) Data are shown as mean±s.e.m. *P<0.05 as determined by t-test. Data are representative of three or more independent experiments.