PMC

Ventral view of the palatal region of wild-type and homozygous embryos at E18.5. Secondary palate (indicated by arrows) was closed in wild-type (A) and Sumo-1^−/− (B) embryos. Histological sections confirm complete palate closure in a Sumo-1 heterozygote (C) and homozygote embryo (D). Arrows indicate the midline of the palate where the two palatal shelves have fused.

Postnatal growth rate of mice wild-type an Sumo-1-null mice. The body weights of male (A) and female (B) littermates were recorded over a 12-month period. There were at least six male and female mice of each genotype. Wild-type, +/+; Sumo-1^−/−, −/−. Mean ± standard error of the mean (SEM) values are shown.

Immunohistochemical analyses of SUMO-1 (A, B, E, and F) and SUMO-2/3 (C, D, G, and H) antigens in the testes of wild-type (A, C, E, and G) and Sumo-1^−/− (B, D, F, and H) mice at 10 days of age (10 days) and in adulthood (adult). XY bodies are depicted by the dark brown spots within the semineferous tubules. Bar, 50 μm.

Sumo-1 and Sumo-2/3 mRNA levels in wild-type and Sumo-1^−/− mice. (A) RNA blot analysis of Sumo-1 mRNA and Sumo-2/3 mRNA levels in the different tissues from adult wild-type (+) and Sumo-1^−/− (-) mice. The bottom panel depicts 28S and 18S rRNAs in the corresponding samples. (B) Quantitative RT-PCR analysis of Sumo-1, Sumo-2, and Sumo-3 mRNA abundance in the testes of adult mice. The values were normalized by Wbscr1 mRNA levels, and the results are expressed as mean ± SEM values of three individual samples. The mean wild-type value was 1.0.

Immunoblot analysis of SUMO-1, SUMO-2 and RanGAP1 proteins in wild-type and Sumo-1^−/− MEFs. (A and B) Immunoblots of lysates from wild-type and Sumo-1^−/− MEFs were generated by using anti-SUMO-1 antibody (A) or anti-SUMO-2 antibody (B). (C and D) MEFs lysates were immunoprecipitated with anti-SUMO-1 antibody (C) or anti-SUMO-2 antibody (D), after which the antibody-antigen complexes were resolved by electrophoresis on a 12% SDS gel and immunoblotted with anti-RanGAP1 antibody. IB, immunoblotting; IP, immunoprecipitation. The molecular mass of RanGAP1 is ∼70 kDa (arrow) and that of sumoylated RanGAP1 is ∼90 kDa (arrowhead).

Absence of SUMO-1-conjugated proteins and free SUMO-1 in MEF and testis lysates of wild-type and Sumo-1-null mice. Proteins in cell lysates were resolved by electrophoresis on 4 to 20% polyacrylamide gradient gels under denaturing conditions and transferred to nitrocellulose membranes. Panels A to C show immunoblots (IB) with anti-SUMO-1 antibody (A), anti-SUMO-2 antibody (B), and anti-VP16 antibody (C). In panel A, the narrow strip shows the free SUMO-1 region after a 10-fold longer exposure than for the main immunoblot. Anti-β-tubulin IB is shown for comparison in each instance. Asterisks depict the ∼90-kDa band corresponding to sumoylated RanGAP1; arrowheads mark free SUMO-1, and the arrow identifies free SUMO-2.

Adipocyte differentiation of wild-type and Sumo-1^−/− MEFs. Two days after reaching the confluence, the MEFs were induced to differentiate into adipocytes. (A) Cells were stained with Oil Red at 10 days after induction. Adipocyte numbers were counted from eight randomly selected fields from four dishes and were 515 ± 125 (SEM) and 467 ± 23 (SEM) for wild-type and Sumo-null MEFs, respectively. (B) Levels of PPARγ and aP2 mRNAs in adipocytes differentiated for 10 days from wild-type and Sumo-1^−/− MEFs. “Control” refers to MEFs that were cultured in the absence of differentiation medium. The values were normalized by Gapdh mRNA levels, and the results are expressed as mean ± SEM values of three samples.

Targeted disruption of the Sumo-1 gene. (A) Replacement targeting vector to delete exons 3 to 5 of Sumo-1. The approximate locations of PCR primers used to screen for homologous recombinants and genotypes are shown (arrows) with the original and predicated structures of the gene after homologous recombination. The location of probe 3 used in Southern blotting is also depicted. (B) Amino acid sequence of SUMO-1. Color coding refers to the regions encoded by different exons of Sumo-1. The sequence of the 29-amino-acid long peptide that is potentially encoded by the targeted Sumo-1 gene is shown below. (C) Positive ES clones were found to contain homologous recombination of Sumo-1 by PCR screening using the primers Neo1 and SUMO-1,P2 shown in panel A. (D) Genomic DNA was isolated from two wild-type ES clones (WT) and one representative ES clone with homologous recombination of Sumo-1 (KO), digested with HindIII, and analyzed by Southern blotting. The presence of both 3.2- and 8.6-kb bands indicates the presence of homologous recombination. (E) RNA blot hybridization analysis of samples isolated from testes of wild-type (+/+), heterozygous (+/−), and Sumo-1-deficient (−/−) mice with probes specific to Sumo-1 and α-actin mRNA. The Sumo-1 cRNA probe corresponds to nt 104 to 359 of Sumo-1 mRNA and thus extends from 3′ end of exon 1 until 5′ end of exon 5 of the Sumo-1 gene.