Fig. 4.
PKR is required for the inhibition of cell growth by PTEN. PKR+/+ (A) and PKR–/– MEFs (B) were transfected with pcDNA-GFP-zeocinR vector (Zeo control), as well as pcDNA vector bearing either FLAG-PTEN WT cDNA or FLAG-PTEN C124S cDNA in the presence of pcDNA-GFP-zeocinR vector. The pcDNA vector encoding the PTEN inserts was used in 10-, 5-, or 2-fold excess over the control pcDNA vector bearing the zeocin resistance gene. Transfected MEFs were selected in zeocin (400 μg/ml) for 3 weeks, and colonies were fixed in methanol and stained with crystal violet. Plates were scanned and quantification of the clones in the images was performed with Scion Image 4.0.3.2 software. Histograms show quantification of results from three independent experiments; error bars indicate the SD. The group difference was tested by ANOVA (P < 0.0001 for comparisons). Protein extracts (50 μg) from cells harvested 48 hours after transfection were subjected to Western blotting analysis with antibodies against the FLAG epitope and actin. Data shown are representative of three experiments.