Fig. 4
From: Loss-of-function phenotype of a PKCθT219A knockin mouse strain
Mutation of (p)T219 on PKCθ leads to NFAT and NF-κB transactivation defects in activated T cells. a and b, the nuclear extracts of resting and stimulated (overnight) wild-type and PKCθT219A CD4+ T cells were probed for DNA binding to radio-labeled (a) or biotinylated (b) probes containing NFAT (a) and NF-κB (b) binding site sequences, as indicated. One representative EMSA experiment of three is shown. The alpha screen measurement shows the summary of four independent NF-κB DNA binding experiments. Data are shown as means ± SEM (n = 4). Unpaired Students t-test was used for statistics. c, Immunoblots revealed an impaired nuclear import of NFAT and NF-κB transcription factors in activated T219A CD4+ T cells. Nuclear extracts of resting and stimulated (overnight) wild-type and T219A CD4+ were probed with antibodies against NFAT and the NF-κB subunit p50. DNA polymerase served as the loading control. One representative experiment of three is shown. The Gel shift result (EMSA) and nuclear NFAT and p50 protein levels (immunoblot) were quantified by densitometric analysis. Numbers beneath bands indicate changes compared to stimulated wild-type controls that has been set as 100. d, Ca2+ mobilization assay revealed an impaired intracellular Ca2+ influx upon CD3 crosslinking in mature CD3+ from PKCθT219A knockin and PKCθ knockout mice. One representative experiment of three is shown