Figure 4

Agnoprotein inhibits cell proliferation. A375 cells were transfected with empty expression plasmid pRcCMV (CTRL), expression plasmid for agnoprotein (Agno), expression plasmid for PCNA, or expression plasmids for agnoprotein and PCNA, respectively. (A) Cell proliferation was measured by the MTT method 48 hours after transfection. The value obtained for pRcCMV-transfected cells was arbitrary set as 100% and the values for the other cells were related to this. The values are the average of six independent parallels measured in triplicate. The standard deviation is shown and a student’s t-test was performed. Similar results were obtained in a separate experiment. (B) Ectopically expressed agnoprotein and FLAG-tagged PCNA were detected by western blot on lysates of transfected cells using anti-agnoprotein and anti-FLAG-specific antibodies. Lane 1: lysates of cells transfected with empty expression plasmid pRcCMV; lane 2: lysates of cells transfected with pRcCMV-agnoprotein expression plasmid; lane 3: lysates of cells transfected with an expression plasmid encoding FLAG-tagged PCNA; lane 4 lysates of cells co-transfected with expression plasmids for agnoprotein and FLAG-tagged PCNA. To assure equal loading, expression levels of ERK2 in each lysate were determined (bottom panel).