Fig. 4

Expression of truncated Runx1 protein from the Runx1 ^ΔP1:P2TAA allele. a Genotyping of offspring obtained by intercrossing Runx1 ^ΔP1:P2TAA heterozygous mice at four weeks. b Cell lysates prepared from total thymocytes of adult mice of indicated genotypes were immunoblotted with an anti-Runx1 antibody. Serial dilutions, each reduced by half, were prepared from cell lysates from Runx1 ^{ΔP1:P2TAA/ΔP1:P2TAA} mice. The membrane was re-blotted with an anti b-actin antibody as an internal control. One representative image of two experiments. c Semi-quantitative RT-PCR analysis showing similar amounts of P2-Runx1 transcript between Runx1 ^P1N/P1N and Runx1 ^{ΔP1:P2TAA/ΔP1:P2TAA} thymocytes. d Effect of proteasome inhibitor, MG132, on the amount of Runx1 proteins. CD4⁺ T cells prepared from Runx1 ^+/+ and Runx1 ^{ΔP1:P2TAA/ΔP1:P2TAA} mice were treated with 10 μM MG132 proteasome inhibitor for one hour. Numbers at the bottom indicate relative expression level to that of Runx1 ^+/+ cells without MG132 treatment. e Expression of Runx1 protein from three expression vectors harboring different sequences around the translation start site, ATG, TAG and TAA, on P2-Runx1 cDNA. Different amounts (1.0, 0.5, and 0.25 μg) of these vectors were transfected into 293 T cells. Forty-eight hours after transfection, cell lysates were immunoblotted with an anti-Runx1 antibody. Empty vector (Emp.Vec.) and thymocyte lysates were included as references