FIG. 5.

Monitoring double transgene expression. Proteins extracted from transduced PLAP-positive neomycin-resistant NIH 3T3 cells were used to determine the level of expression of each transgene by vector constructs. (A) PLAP enzymatic activities were determined as described in Materials and Methods (53, 84). The mean values of alkaline phosphatase specific activities as well as the standard deviation for each set of experiments are shown. Data are the averages of values from three independent experiments. (B) Ten micrograms of total protein was loaded per lane and subjected to SDS–15% PAGE. Proteins were transferred to a polyvinylidene difluoride membrane and probed with a rabbit anti-neomycin phosphotransferase II antibody. The membrane was then incubated with a biotinylated anti-rabbit immunoglobulin G antibody and an avidin-peroxidase solution and, finally, developed by enhanced chemiluminescence. Lane 1, negative control (protein extract from nontransduced NIH 3T3 cells); lanes 2 through 6, protein extracts from cells transduced with the different retroviral vectors. The positions of molecular mass standards are shown on the left.