Phosphorylated JNK (p-JNK) can be found in the nucleus as well as in the cytoplasm. Following a 6-day primary culture, anergic Th1 cells contained p21Cip1 in both cytoplasmic and nuclear fractions, although there was more p21Cip1 in the cytoplasmic fraction than the nuclear fraction (Fig. 3). In contrast, control Th1 cells contained little p21Cip1 in either fraction at the end of the 6-day primary culture. The presence of U1, a small nuclear ribonuclear protein of molecular weight 70 000 (SnRNP 70) in the nuclear fractions from both anergic and control Th1 cells
confirmed efficient nuclear fractionation. The mechanistic significance of the p21Cip1 detected in the anergic Th1 cells was examined in the next series of experiments. As p21Cip1 was found in both cytoplasm and nucleus of anergic Th1 cells, www.selleckchem.com/products/pembrolizumab.html all three interaction partners
of p21Cip1 known to mediate cell cycle inhibition, Palbociclib in vitro namely cdk, PCNA and JNK, were examined for their association with p21Cip1 in these cells. p21Cip1 was first examined for its ability to bind to cdk. Cdk2, cdk4 and cdk6 were examined for coprecipitation with p21Cip1 in anergic and control Th1 cells following antigen restimulation. The restimulation period was extended to 36 hr to allow enough time for the control Th1 cells to up-regulate p21Cip1. The upper blots demonstrated that the cdk were immunoprecipitated efficiently such that very little of the relevant cdk remained in the supernatant
(Fig. 4). It was noted that anergic Th1 cells contained little cdk2, probably because of the requirement for IL-2 in cdk2 up-regulation. As expected, p21Cip1 was found associated with cdk2, cdk4 and cdk6 in control Th1 cells 36 hr after antigen stimulation. However, p21Cip1 in the anergic Th1 cells did not demonstrate an PAK5 increased association with cdk compared with the control Th1 cells. Proliferative unresponsiveness in the anergic Th1 cells therefore could not be attributed to preferential p21Cip1 interaction with cdk. Considering the possibility that p21Cip1 interaction with cdk could have taken place in the anergic Th1 cells earlier in the secondary cultures before p21Cip1 was up-regulated in the control cells, the p21Cip1–cdk interactions were examined in lysates obtained from anergic Th1 cells restimulated for only 2 hr. Control lysates were obtained from Th1 cells that were restimulated for 24 hr to up-regulate sufficient p21Cip1 levels for detection (Fig. 5a). p21Cip1 was immunoprecipitated from the lysates and examined for binding partners. All experimental groups, including 24-hr-stimulated control Th1 cells, anergic Th1 cells before restimulation and anergic Th1cells following 2 hr of restimulation, contained p21Cip1 that was immunoprecipitated successfully from all three lysates (Fig. 5b).