Improved Flp-In INS-1 cell lines. (A) Schematic presentation of the vector construct to generate Flp-In INS-1 cell lines conditionally expressing HNF4α under control of the 5'-deleted CMV promoter (left) or the P2 promoter (P2/-2200 from ) from the human HNF4α gene (right). Both promoters are tetracycline inducible due to the operator sites (2*TetO2) upstream of the transcription start site and the constitutively expressed tetracycline repressor (TetR) . (B) Western blotting of whole cell extracts of the indicated Flp-In INS-1 cell lines with the HNF4α-antibody C-19 (Santa Cruz) to detect the HNF4α2 splice variant expressed from the full length or 5' deleted CMV promoter. Cells were cultured without (- tet) or with (+ tet) 50 ng/ml tetracycline for 24 h. 18 μg of total protein were loaded per lane and the signal for the exogenous (exo) as well as endogenous (endo) HNF4α is marked. Since the Flp-In INS-1 cell line #1-1.2 used previously  has lost its glucose induced insulin secretion (data not shown), we used now the cell line #5-3.19 that retains glucose sensitivity (data not shown) and that shows an identical expression level of endogenous HNF4α (lanes 1 and 2). The observation of differential characteristics in the INS-1 cells has been observed in our initial study  and reflects a property typical of the INS-1 cell line .