Id1 expression is increased after inducible expression of either BCR-ABL or FLT3-ITD. (A) TonB/BCR-ABL or TonB/FLT3-ITD cells were deprived of IL-3 for 15 hours before adding doxycycline (2 μg/mL). Whole cell lysates were harvested at the time point indicated and analyzed by Western blot using either c-Abl or FLT3 antibody (top panel). The membranes were reblotted with a Phospho-Stat5 (Tyr694) antibody (bottom panel). (B) Total RNA and whole cell lysates were harvested at 48 hours after addition of doxycycline (2 μg/mL). Real-time quantitative RT-PCR analysis of Id1 expression in inducible cell lines using Id1 specific primers (left). The results were normalized against the expression level of GAPDH. The corresponding Western blot analysis using Id1 antibody is listed on the right. The membrane was stripped and reblotted with RACK1 antibody as loading controls. (C) Total RNA and whole cell lysates were harvested at 48 hours after addition of doxycycline (2 μg/mL). Real-time quantitative RT-PCR analysis of Id2 expression in inducible cell lines using Id2 specific primers (left). The results were normalized against the expression level of GAPDH. The corresponding Western blot analysis using Id2 antibody is listed on the right. The membrane was stripped and reblotted with RACK1 antibody as loading control. (D) Total RNA was isolated from spleens of mice that developed myeloproliferative disease. Real-time quantitative RT-PCR was performed using Id1-specific primers. The results were normalized against the expression level of GAPDH. Total RNA from normal spleen was used as a negative control. (E) Total RNA was isolated from FACS-sorted GFP-positive primary murine bone marrow cells transduced with retroviral vectors coexpressing GFP alone, or GFP with either BCR-ABL or FLT3-ITD.