Figure 2.

Expression of AAV2 Cap and Rep proteins from natural promoters. Transformed RSY12 cells were grown in liquid selective medium until cultures reached different densities determining different growth phases (early → late log) indicated on the top. Equal amounts of total cell lysate, extract 1 and 2 (~50 μg each), obtained from 2 × 10⁸ cells at each phase, were analyzed by Western blot analysis with antibodies indicated on the left: mAb B1 to detect Cap proteins (A and C), mAb 303.9 to detect Rep proteins (B) and mAb Anti-3PGK to detect constitutive yeast protein PGK (Phospho-Glicerate-Kinase). (A): VP3 was the only capsid protein detected in the samples from YEplacp40Cap transformed cells;thehighest level was detected in the mid-log phase extracts. (B): All four Rep proteins were detected in the samples from YEplacRepCap transformed cells; the highest level was obtained in the late-log phase extracts. (C): late-log phase extracts from YEplacRepCap cells were also analyzed for Cap protein expression and compared with late-log phase extracts from YEplacp40Cap cells; only VP3 was detected in both samples. Denatured, 293 T-cell-derived, AAV2 capsids were used as positive control (+ control) for defining VPs (A, C).