FIG. 3.

NL63 PLP2 has global deISGylating activity in cultured cells. HEK293 cells were transfected with pcDNA3-myc6-mISG15, pcDNA3-Ube1L, and pcDNA3-UbcH8 plus indicated amounts of the PLP2 expression construct (A) or PLP2 expression construct and the corresponding catalytic mutants (B). To ensure that the total amount (2 μg/transfection) of plasmids for transfection was equal under every condition, empty vector pcDNA3.1/V5-HisB (v) was used to standardize the total amount of DNA. After 30 h, cells were harvested, and cell lysates were analyzed for ISG-conjugated proteins by Western blotting with monoclonal anti-myc antibody. Expression levels of V5-tagged PLP2 and mutant enzymes were detected with anti-V5 antibody. Actin was detected with antiactin MAb antibody as a protein loading control.