JCS038570 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. X-gal-stained embryos from XA024 line. The genotypes are below each embryo; wt, wild type; het, heterozygous; hmz, homozygous. (A) E11.5 embryos taken from the same litter. Homozygote shows more intense staining than heterozygote. (B) E14.5 embryos taken from the same litter. Homozygote shows more intense staining than heterozygote and no phenotypic abnormalities.

• Supplemental Figure S2 -

  Fig. S2. Truncated SUMO1 is not stably expressed in HEK293 cells. (A) Immunoblot analysis of bacterially expressed His-tagged SUMO1, either full length (lane 1) or internally deleted as in XA024 (lane 2), revealed equally efficiently recognition by anti-SUMO1 antibody of both constructs. (B) Pyo-epitope-tagged SUMO1 expression constructs, either full length (lanes 1 and 3) or internally deleted (lanes 2 and 4), were transiently transfected into HEK293 cells along with a control EGFP expression vector. An equivalent efficiency of transfection was estimated by GFP fluorescence (not shown). Whole cell extracts were analyzed by immunoblotting with anti-SUMO1 (lanes 1 and 2; 4-12% gel) or anti-pyo antibodies (lanes 3 and 4; 18% gel). Whereas expression of full length pyo-SUMO1 was easily detected with either antisera (lanes 1 and 3), no expression of truncated pyo-SUMO1 could be detected (lanes 2 and 4). Anti-MAPK1 antibody was used for estimating protein loading for lanes 1 and 2; non-specific bands detected by anti-pyo (asterisks) were used to estimate protein loading in lanes 3 and 4. There is an apparent effect on endogenous (endg.) SUMO1 expression in cells transfected with the truncated pyo-SUMO1.

• Supplemental Figure S3 -

  Fig. S3. Structure of gene-trap insertion in RRQ016 line. Upper, schematic representation of exon 1, intron 1 and exon 2, of the wild-type SUMO1 gene. The position of the insertion and of the region presumably duplicated is shown. Middle, structure of the gene trap allele showing inserted vector. The precise 3′ junction is unknown. Lower, structure of the gene-trap vector. A significant amount at the 5′ end was lost in the insertion event.

• Supplemental Figure S4 -

  Fig. S4. X-gal staining of RRQ016 embryos. The presumed genotypes based on staining intensity are shown below each embryo; wt, wild type; het, heterozygous; hmz, homozygous. The E15.5 embryos were taken from the same litter. Presumed homozygote shows more intense staining than heterozygote and displays no phenotypic abnormalities.

• Supplemental Figure S5 -

  Fig. S5. Immunoblot analysis of RRQ016 embryos. Whole embryo extracts from four litters collected at two developmental stages were analyzed anti-SUMO1 antibody (top panels). The same blots were then reanalyzed with anti-RanGAP1 antibody (middle panels) and anti-β-actin antibody as a loading control (lower panels). The genotypes are shown above each lane; +, β-geo positive; −, β-geo negative. There is no loss of sumoylation in any β-geo-positive embryo. Samples from E8.5 litters represent entire embryo. Individuals with lower levels were developmentally earlier and thus smaller and gave lower protein yield.

• Supplemental Table S1 -

  Table S1. Determination of PML NB numbers in individual cell nuclei. Each nucleus was semi-automatically delineated and PML NBs were automatically detected from the confocal image that sliced approximately through the center of each nucleus. Values of manually set parameters in the automatic spot detection algorithm were the same for all nuclei. (A) Number of PML NBs detected per nuclear area for each nucleus from three images of wild type (WT) cells, three images of heterozygous (Het) cells and four images of homozygous (Hmz) cells. (B) Means and sample s.d. of the number of detected PML NBs per nucleus for each of the cell types. (C) Student’s t-test results reporting significantly fewer PML NBs per nucleus in the homozygous cells versus the heterozygous and wild-type cells. (D) Means and sample s.d. of the ratio of PML NBs to unit area of nuclei for each of the cell types. (E) Student’s t-test results reporting significantly fewer PML NBs per unit area of nuclei in the homozygous cells versus the heterozygous and wild-type cells.