Fig. 3.

Over-expression of CHOP, but not its N-terminal deletion mutant, promotes IBV-induced apoptosis. (A) H1299 cells were transfected with different amount of FLAG-tag CHOP plasmid or empty vector. At 24 h post transfection, cells were infected with IBV or incubated with mock lysate for 22 h. Cell lysates were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membrane for western blotting using antibodies against FLAG-tag, IBV N protein and the apoptosis marker poly (ADP-ribose) polymerase (PARP). The percentages of PARP cleavage (intensity of cleavage band [Cl] divided by total intensities of full-length [FL] and cleavage bands) were determined and indicated below. The β-tubulin protein was used as loading control. The presented blot is one representative blot from three independent experiments. (B) Schematic diagram showing the known functional domains of CHOP and the two N-terminal deletion mutants (ΔN36 and ΔN70) used in (C). Amino acid 10–18 have been shown to interact with TRIB3. Serine 79 and 82 are phosphorylated by p38, while Leucine 134 and 141 are responsible for DNA binding of the bZIP domain. (C) H1299 cells were transfected with FLAG-tag wild type CHOP or N-terminal deletion mutants. At 24 h post transfection, cells were infected with IBV or incubated with mock lysate. Cell lysates were harvest and subjected to SDS-PAGE and western blot analysis as in (A). Percentage of PARP cleavage is determined as in (A) and indicated below. The β-actin protein was used as loading control. The presented blot is one representative blot from three independent experiments.