Figure 4.
Phenserine regulation of βAPP protein levels in U373 MG astrocytoma cells is conferred through the action of the βAPP–mRNA 5′ UTR. U373 MG astrocytoma cells were transfected with 3 μg of pSV2 (APP) CAT plasmid or the parental vector pSV2 CAT. Each set of transfection plates was left unstimulated or treated with 50 μM of phenserine for the experimental times listed. (A) CAT activity was assessed from lysates of transfected cells treated with (−)-phenserine for 0, 4, and 8 h, and 50 μg from each sample was measured in duplicate for each assay. Quantitation of the fold stimulus of CAT gene activity conferred by the 5′ UTR βAPP–mRNA was measured. ELISA readings of CAT expression were measured at 405 nm. (B) Phenserine does not affect the steady-state levels of βAPP–mRNA levels. Ten micrograms of RNA isolated from untransfected and transfected cells treated with (−)-phenserine for 0, 1, 4, 24, and 48 h was analyzed by Northern blot. Phosphoimager analysis revealed steady-state expression of βAPP–mRNA in each sample. The filter was stripped and rehybridized with a labeled human actin probe to standardize the loading differences in individual samples.