Repression of transgene expression in vitro with doxycycline. (A) Cryopreserved spleen cells from affected animals were incubated 80 hours with doxycycline (Dox) at the indicated concentrations. Total RNA was extracted, DNase-treated and NRAS RT-PCR was performed with (+RT) and without (-RT) reverse transcriptase. GAPDH RT-PCR was performed on the same samples to verify integrity and loading. (B) Cultured cells were split, and half were treated with doxycycline for 24 hours. Treatment was continued while cells were serum starved overnight and then stimulated with 5 ng/mL SCF for the times indicated. Lysates were harvested on ice, and Western blot analysis was performed for p-ERK. The blot was stripped and reprobed for total ERK1 to verify equivalent loading.