Pax5−/− huPax5 pro-/pre-B-cell clone 4 expresses CD19 and down-regulates Flt3 surface expression on doxycycline-induced huPax5 expression in vitro. (A) The retroviral huPax5-TetON-expression system. huPax5 will be transcribed after binding of the doxycycline-dependent reverse transactivator rtTA-S2 to the transactivator-dependent TRE-element containing a pCMVmin. (B) Quantitative RT-PCR for huPax5 mRNA levels in doxycycline-induced Pax5−/− huPax5 pro-/pre-B-cell clone 4 before and 3 days after doxycycline administration (1000 ng/mL) in vitro. Each bar represents the mean ± SEM (error bars) of 3 individual experiments. (C) Western blot analysis with a Pax5-specific antibody of whole cellular lysates of Pax5−/− huPax5 pro-/pre-B-cell clone 4 before and 3 days after doxycycline administration (1000 ng/mL) in vitro in comparison to wild-type pre-B-I cells and Pax5−/− rtTA pro-/pre-B cells. β-actin–specific antibody was used as a loading control. (D) Monitoring of huPax5-dependent CD19 and Flt3 expression by FACS analysis of doxycycline-induced Pax5−/− huPax5 pro-/pre-B-cell clone 4. The numbers represent percentages of Flt3+ or CD19+ pro-/pre-B cells before and 3 days after doxycycline administration (1000 ng/mL) in vitro. Data are representative for 3 individual experiments.
