Fig. 1

Disruption of the GAREM2 gene in mice. a Schematic representation of the GAREM2 targeting vector, the mouse GAREM2 gene, the targeted allele, and the deleted allele. A neomycin-resistance gene with a Pgk1 promoter and polyadenylation signal (PrNeopA), FRT sequences, and the loxP sequences are shown by open boxes, dark triangles, and filled triangles, respectively. PrDT-ApA is a diphtheria toxin A fragment gene with a MC1 promoter and rabbit β-globin gene poly A signal for negative selection [50 = 30]. Positions of probes used for Southern blotting analyses with HincII are also shown. The targeted alleles after Cre-recombination are shown at the bottom. b Southern blotting analysis of mouse tail genomic DNA of WT and chimeric mouse with the targeted allele. The 8.1 kbp band corresponds to the WT allele, and the 10.2 kbp band corresponds to the targeted allele involving the neomycin gene. c Genotyping mice by PCR after mating with CAG-Cre transgenic mouse. Mice or MEF were routinely genotyped by PCR using two primers {P1 and P2}. The P1/P2 primer set yielded the 1242 bp product from the WT and the 289 bp product from the mutant allele. d Characterization of mouse GAREM2 protein. Detection of endogenous mouse GAREM2 with anti-GAREM2 antibody. Protein expression of GAREM2 (upper), and β-actin (lower) in mouse tissues. Endogenous GAREM2 in the WT lysate is indicated by an arrow. Total cell lysate (20 μg) of heart, brain, and Liver from male WT and KO mice at 8 weeks of age were separated by SDS-PAGE and analyzed by immunoblotting with each antibody (right panels). e Expression of GAREM2 in nine brain regions. The nine brain regions were dissected and sampled from adult WT (left) and KO (right) male mice, and the total cell lysates were separated by SDS-PAGE and immunoblotted with an β-actin loading control (bottom panel). FLAG59Bfull indicates the total cell lysate of COS-7 cells transfected with FLAG-tagged GAREM2, which was immunoprecipitated with anti-FLAG antibody and used as a positive control