Making of a HeT-A Gag-FLAG stable cell line.
(A) Line diagram of the PMK33 vector showing important genetic markers with HeT-A Gag ORF1 cloned at the SmaI restriction site and a 3x FLAG tag at the C-terminus. (B) Expression of HeT-A Gag-FLAG in S2 cell lines as analyzed by PCR and immunoblot. GAPDH and actin serve as internal controls for PCR and immunoblots respectively. (C) Stability of expressed HeT-A Gag-FLAG protein in S2 cells. Cells were seeded in a T-25 flask and induced with 500 µM CuSO4. 1.0 ml cells were collected each day for a total of 6 days and analyzed by immunoblotting. (D) HeT-A Gag-FLAG is targeted to the nucleus. Stably transfected S2 cells were induced with CuSO4 and 48 hr post-induction cytoplasmic and nuclear fractions were analyzed by immunoblot. (E) HeT-A Gag-FLAG is only detected in stably transfected S2 cells and not in S2 cells. Cytoplasmic, nuclear, and nuclei fractions from both S2 and stably transfected S2 cells were analyzed by immunoblot (1 = cytosolic, 2 = nuclear, and 3 = nuclei).