Figure 3
Relative transcript copy numbers and Western blot of amplified suspension cell pools. Exponentially growing cells were harvested from shake flask cultures of the 50 nM and 300 nM MTX amplified suspension cell pools from each vector set. (A) First-strand cDNA from each sample were analyzed by qPCR. Threshold cycle (Ct) data were analyzed using the ΔΔCt method using pAID as sample reference and β-actin as normalizer. Relative transcript copy numbers were calculated as 2ΔΔCt. Standard deviations from technical triplicates were determined to be lesser than 10% of the relative values. The relative specific productivity qp was obtained by normalizing exponential qp to that of pAID at the respective MTX concentrations. (B) 20 μg of total protein from each sample were resolved by SDS-PAGE, transferred onto a PVDF membrane, and probed with primary antibodies against dhfr and β-actin.