Generation of MLF1-IP KO mice.

<p><i>A. Targeting strategy for generating MLF1-IP KO mice</i> The red square on the 5′ side of the mouse MLF1-IP WT genome represents the sequence used as a probe in Southern blotting for genotyping. <i>B. Genotyping of MLF1-IP mutant mice</i> Tail DNA was digested with NcoI, and analyzed by Southern blotting (left panel), with a 5′ probe whose location is indicated in A (red square). A 10-kb band representing the WT allele and a 6.8-kb band representing the recombinant allele are indicated by arrows. Ear lobe DNA sample was analyzed by PCR for routine genotyping (right panel). A 588-bp band representing the WT allele and a 202-bp band representing the recombinant allele are indicated by arrows. <i>C. MLF1-IP mRNA expression in MLF1-IP WT, HET and KO embryos</i> mRNA levels from e7.5 WT, HET and KO embryos were analyzed by RT-qPCR. The results are expressed as ratios of MLF1-IP versus β-actin signals with means±SD indicated. <i>D. MLF1-IP mRNA expression in WT and HET T cells upon activation.</i> T cells from WT and HET spleens were stimulated by solid-phase anti-CD3 mAb and anti-CD28 mAb (0.5 µg/ml and 4 µg/ml respectively for coating) for 0, 24, 48 or 72 h, and their MLF1-IP mRNA levels were quantified by RT-qPCR. The results are expressed as ratios of MLF1-IP versus β-actin signals with means±SD indicated.