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Fig. 4. Both TNFR1- and TNFR2-mediated apoptosis in PC60 R1R2 cells is FADD dependent. (A) PC60 R1R2 were electroporated with: pUT651, a ß-gal expression plasmid, plus pCDNAI (control) or plus pCDNAFADD-DN, encoding dominant negative FADD (80-205). 48 hours after transfection cells were lysed and 150 µg of protein was submitted to immunoblotting using a monoclonal FADD-specific antibody. (B) 24 hours after electroporation, cells were stimulated overnight either with 500 ng/ml R32WS86T, 500 ng/ml D143NA145R or 100 ng/ml hTNF. Subsequently, cells were stained with X-gal and ß-gal-positive blue cells were examined microscopically for apoptotic features, such as membrane blebbing and nuclear changes, after which the percentage of induced apoptotic cells was determined (500 blue cells were counted in each measurement).
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