Fig 7.

ORF2 reduces the steady-state levels of Notch3. (A) Neuro-2A cells were transfected with the designated plasmids and then collected and processed for Western blot analysis. Cultures were treated with 100 μM CHX for 1 or 2 h at 40 h after transfection. To inhibit proteasome activity, cells were treated with lactacystin (15 μM) 10 h prior to CHX treatment. (B) The 2A/ORF2 plasmid contains ORF2 cloned such that there is a one-nucleotide frameshift downstream of the N-terminal Flag epitope. The 2B/ORF2 plasmid contains ORF2 in frame with the N-terminal Flag epitope. The Western blots were probed with a Flag-specific monoclonal antibody or an ORF2-specific peptide antibody. (C, D) The effects of the respective mutant ORF2 constructs on steady-state Notch3 protein levels were examined. Neuro-2A cells were cotransfected with ORF2 or the designated mutant ORF2 constructs and a plasmid expressing Notch3. Cultures were treated with CHX as described above. A mouse anti-Flag antibody was used to detect WT and mutant ORF2, while Notch3 was detected by using a rabbit anti-Notch3 antibody. β-Actin protein levels were determined to confirm that equal amounts of protein were loaded in each lane. The molecular weights (10³) of the respective bands are shown to the left of each Western blot.