P45 reduces FasL-induced cell death signalling.

A. Brain protein extracts from controls and the Thy1-p45 transgenic mice were immunoprecipitated with an anti-p45 or an anti-FADD antibody and followed by immunoblotting with an anti-FADD or an anti-p45 antibody, respectively. The efficiency of immunoprecipitation and expression levels of p45 and FADD were validated with appropriate antibodies as indicated. B. MCF7-hFAS-CTL and MCF7-hFAS-p45 cells were treated with FasL (10 ng/ml) for 0 (–) or 30 (+) min. The lysates were immunoprecipitated with an anti-Fas antibody and followed by immunoblotting with an anti-caspase-8 (Casp-8) antibody. The recruitment of Casp-8 to the DISC is markedly reduced by p45, but not FADD. The efficiency of immunoprecipitation and expression levels of Pro-casp-8, FADD, Fas and p45 were validated with appropriate antibodies as indicated. (C, D) MCF7-hFAS-CTL and MCF7-hFAS-p45 cells were treated with FasL (10 ng/ml) for 0–360 min. The lysates were immunoblotted with an anti-caspase-8 or an anti-PARP antibody. The cleavage of both proteins is markedly attenuated in p45-expressing cells. An anti-actin antibody was used for protein loading control. Closed arrowhead, full-length protein; open arrowhead, cleaved products. (E) FasL- or anti-Fas antibody CH11-treated cells were stained with DAPI to detect shrunken nuclei, indicative of cell death. P45-expressing cells significantly reduced cell death induced by either treatment. ***P