Fig. 2. Proteolysis of overexpressed POM121-GFP and endogenous POM121 during apoptosis. Monolayer cultures of stably transfected neuroblastoma SH-SY5Y cells (GI97-4 cells), stably transfected PC-12 cells or BRL cells, were treated with staurosporine for different times. (A) Fluorescence micrographs showing equatorial sections of GI97-4 cells (a,b,e,f) and BRL cells (c,d,g,h), treated for 15 hours. Pairs of images of control (-STSP) and treated (+STSP) cells were acquired and processed identically. The GFP fluorescence (a,e) and the immunostaining using anti-POM121 antibodies (c,g) are shown to the left of the corresponding images of DNA staining (b,d,f,h). Bar, 20 µm. (B) Western blot analysis of POM121 and POM121-GFP. Treated (+STSP) and control (-STSP) cells were solubilized in sample buffer after 8 hours of treatment and subjected to SDS-PAGE and western blotting using anti-POM121 or anti-GFP antibodies. In apoptotic cells, overexpressed POM121-GFP (
) as well as endogenous POM121 () decreased. (C) Western blot analysis of overexpressed POM121-GFP () and endogenous POM121 () in stably transfected PC12 cells undergoing apoptosis (+STSP). After 8 hours of treatment 56% of the overexpressed and 47% of the endogenous protein remained. After 16 hours the corresponding figures were 14% and 17%, respectively. Data are from one representative experiment.
