Figure 2
BACH2-mediated repression of AP-1 driven luciferase expression using the inducible reporter system. (a) Inducible expression system. Tetracycline repressor (TR) protein in its active form (indicated as a circle) binds to the TetO2 sequence upstream of BACH2 cDNA subcloned into the pcDNA4/BACH2 vector inhibiting transcription of BACH2. Tetracycline (Tet) addition changes the conformation and inactivates the tetracycline repressor (TR) protein (indicated as a square), which is subsequently not able to bind to TetO2 sequence, allowing BACH2 transcription to commence. (b) Western blot for indicated proteins of total lysates isolated from the clonally derived inducible-BACH2 and control reporter cell lines with or without tetracycline treatment. (c) Luciferase activity in the inducible-BACH2 and control reporter lines after 6 h PMA/ionomycin stimulation with or without pre-treatment with tetracycline (1 μg/ml). Unpaired two-tailed Student’s t test (c). Data are representative of 3 independent experiments with 3 culture replicates per condition. Bars and error represent mean (SD); ns not significant; ***P < 0.001; ****P < 0.0001.
