Loss-of-function phenotype of a PKCθT219A knockin mouse strain

摘要

T219A mutation does not alter PKCθ mRNA expression and protein stability. Scheme depicting generation of mutated phosphosite (p) Thr-219. The T219A mutation was biochemically confirmed by immunoblot with lysates of unstimulated and phorbol ester (PDBu) stimulated wild-type and T219A CD3 T cells using our specific (p) Thr-219 PKCθ antibody [David Biotech] for immunoprecipitation and subsequent immunoblot with panPKCθ. Phospho-Erk1/2 staining in the whole cell extract was used to control successful stimulation. The T219A mutation did not alter PKCθ mRNA expression and/or protein stability as verified by RT-PCR and immunoblot (showing the whole cell lysates from two independent experiments, referred as 1 and 2) of unstimulated and CD3/CD28 activated CD3 T cells. RT-PCR data summarizing the results of 3 independent experiments ± SEM are shown. Differentiation of naïve CD4 cells into the iTreg subset was not affected in the knockin mice. Naïve CD4 T cells isolated from wild-type and mice were differentiated in vitro under neutral conditions (“TH0”: CD3/CD28 only) and iTreg-inducing conditions (IL-2/TGF-β with blocking antibodies against IL-4, IL-12 and IFN-γ) and analyzed for Foxp3 expression by qRT-PCR on day 3 of culture. The house keeping gene gapdh was used for normalization. Data are shown as means ± SEM (  = 5). The suppressive capacity of wild-type and T219A CD4 CD25 nTreg cells was analyzed in co-cultures with CFSE-labeled CD25 CD4 T cells (Tresp) stimulated with APCs and anti-CD3 antibodies. Bar graphs summarizing results of 3 independent experiments are shown. Data are shown as means ± SEM (  = 3)