<p><b>A.</b> Endogenous GTPCH1 binds polyubiquitin in mouse endothelial cells (SVEC4-10). Mouse endothelial cells were grown to confluence and cell lysates were incubated with UIM-agarose for ubiquitin affinity precipitation (AP). The interaction of GTPCH1 with polyubiquitin chains was analyzed by immunoblotting. The black triangle (▸) indicates the bound GTPCH1 by polyubiquitin chains. Control beads without UIM were used as a negative control. <b>B.</b> Co-immunoprecipitation of GTPCH1 with polyubiquitin in GTPCH1 overexpressed HEK293 cells. FLAG-GCH1 plasmids or empty vectors were transfected into HEK293 cells for 2 days. Cell lysates were incubated with the ubiquitin antibody and immunoprecipitated using protein A/G beads. The asterisk (*) indicates IgG bands. HEK293 transfected with empty vectors were used as a negative control. <b>C.</b> Polyubiquitin chains were co-immunoprecipitated with GTPCH1. HEK293 cell lysates expressing FLAG-GTPCH1 or control vector were incubated with anti-FLAG resin. Proteins that co-immunoprecipitated with FLAG-GTPCH1 were analyzed by immunoblotting using the indicated antibodies. The blot shown is representative of five independent experiments.</p
