Fig. 4. Immunoblot analysis of RanGAP1 and PML sumoylation. (A,B) Whole-cell extracts (WCE) from embryos are shown in left panels, and immunoprecipitation (IP) with anti-RanGAP1 antibody shown in right panels. The genotypes are shown below lanes; hmz, homozygous; wt, wild type; het, heterozygous. There is less sumoylated form in the homozygote both in WCE and IP upon analysis with anti-RanGAP1 antibody (A). IP shows less sumoylated form in the heterozygote and none in homozygote analysis with anti-SUMO1 antibody (B). (C) Analysis with anti-SUMO2 antibody. The immunoprecipitation shows more sumoylated form in the homozygotes compared to the wild type and heterozygotes. The whole cell extract shows an increase in average molecular mass of SUMO2/3 high molecular mass conjugates in homozygotes. (D) Whole cell extracts (WCE) from MEFs immunoblotted with anti-PML antibody (left). Reblotting with anti-MAPK (lower panel) shows equivalent loading of heterozygous and homozygous samples but less protein loaded for the wild-type sample. Therefore, there is less total PML in the homozygote. Immunoprecipitation (IP) of extracts with anti-PML antibody is shown in the middle and right panels. Immunoblotting with anti-PML antibody is shown in the middle panel and with anti-SUMO2 in the right panel. There is less SUMO2/3 conjugation of PML in MEFs derived from XA024 homozygotes.