Apoptosis is a cell death process that removes toxic and/or useless cells during mammalian development. The apoptotic process is accompanied by shrinkage and fragmentation of the cells and nuclei and degradation of the chromosomal DNA into nucleosomal units. DNA fragmentation factor (DFF) is a heterodimeric protein of 40-kD (DFFB) and 45-kD (DFFA) subunits. DFFA is the substrate for caspase-3 and triggers DNA fragmentation during apoptosis. DFF becomes activated when DFFA is cleaved by caspase-3. The cleaved fragments of DFFA dissociate from DFFB, the active component of DFF. DFFB has been found to trigger both DNA fragmentation and chromatin condensation during apoptosis. Two alternatively spliced transcript variants encoding distinct isoforms have been found for this gene.

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Validation of DFFA knockdown using flow cytometry. Wild-type(WT, Blue) and knockdown(KD, Green) HeLa cells were stained with DFFA antibody and analyzed using BD flow cytometer.

Flow cytometric analysis of DFFA expression in HeLa cells using DFFA antibody . Green, isotype control; red, DFFA.

Immunocytochemical staining of Hela cells with DFFA antibody . Nuclei were stained blue with DAPI; DFFA was stained magenta with Alexa Fluor® 647. Images were taken using Leica stellaris 5. Protein abundance based on laser Intensity and smart gain: High. Scale bar, 20 μm.

Western blotting analysis using DFFA antibody . Total cell lysates (30 μg) from various cell lines were loaded and separated by SDS-PAGE. The blot was incubated with DFFA antibody and HRP-conjugated goat anti-rabbit secondary antibody respectively. Image was developed using FeQ™ ECL Substrate Kit . DFFA, DNA fragmentation factor subunit alpha.

Western blotting analysis using DFFA antibody . DFFA expression in wild-type (WT) and DFFA shRNA knockdown (KD) HeLa cells with 20 μg of total cell lysates. Hsp90 α serves as a loading control. The blot was incubated with DFFA antibody and HRP-conjugated goat anti-rabbit secondary antibody respectively. Image was developed using NaQ™ ECL Substrate Kit .

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