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Fig. 2. HnRNP C1/C2 are translocated to cytoplasm in PMA-induced pro-apoptotic cells in a manner dependent on ROCK-mediated cytoskeleton rearrangement. (A) D2 cells were treated without (NT) or with 32.4 nM PMA in serum-containing medium on regular dishes. Cells remained in suspension (SUS) and attached to the culture dish (ATT) were harvested at the time indicated. Whole cell lysates, cytosolic and nuclear fractions were prepared and separated by SDS-PAGE, followed by western blot analysis with antibodies against hnRNP C1/C2, Sp1 and TK1. (B) D2 cells treated with 32.4 nM PMA as described above for 4 hour were fixed and incubated with antibody against hnRNP C1/C2, followed by visualization with TRITC-conjugated anti-goat antibody. A parallel PMA-induced suspension culture with 50 µM zVAD-fmk incubation was subjected to the same analysis. Nuclei were stained with DAPI. Scale bar: 8 µm. (C) D2 cells were incubated in serum-free medium on hydrogel-coated dishes and treated with 32.4 nM PMA for 4 hours in the presence of reagents as indicated (0.5 µM latruculin B, LB; 20 µM zDEVD-fmk; 20 µM Y27632; 50 µg/ml cycloheximide, CHX; 5 µg/ml actinomycin D, Act D). (D) D2 cells were treated as (C) in the presence of 50 µM blabbistatin for 4 hours. The cytosolic and nuclear fractions were prepared for Western blot analysis as described above.
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