Western blot analysis of INS-1 cell extracts. Whole cell extracts of the indicated Flp-In INS-1 cell lines were analyzed by Western blotting using the HNF4α-(C-19)-antibody (Santa Cruz) recognizing P1 as well as P2 promoter derived proteins. The cell line INS-1 HNF4α2  was cultured without (-) or with (+) 50 ng/ml tetracycline for 24 h. 20 μg of total protein were loaded. For the induced cell line (+), different amounts of whole cell proteins were used to estimate the fold induction of the transgene. The exogenous HNF4α protein was cloned C-terminally to six myc tag repeats. Therefore, the myc-HNF4α protein has an increased molecular mass of approximately 10 kDa in comparison to the endogenous protein. This results in a considerable shift in the western blot. The unexpected higher mobility of some minor bands of the exogenous HNF4α proteins possibly reflects degradation products.