Quantification of EP24.15 overexpression in HEK293 cells in Experiment
2. HEK293 cells were transiently transfected with empty pShooter
(mock-transfected cells) or with pShooter coding for EP24.15
(EP24.15-transfected cells). A, proteins from crude cell extract
(62.5 μg from mock cells and 5 μg from cells transfected with EP24.15)
were separated by SDS-PAGE on an 8% polyacrylamide gel and transferred to
nitrocellulose membranes, which were incubated with specific rabbit antiserum
against EP24.15 (1:3000). After incubation with an anti-rabbit IgG-horseradish
peroxidase-conjugated secondary antibody (1:3000), the immunoreactive bands
were visualized by chemiluminescence. B, EP24.15 enzymatic activity
was determined in triplicate using a continuous assay with a quenched
fluorescent substrate as described under “Experimental
Procedures.” EP24.15 activity was about six times greater in EP24.15
overexpressing cells then in the mock control group. ***, p <
0.001, statistically different from mock control group using Student's
t test.