Fig. 3

NEDD4 mediates mono- and polyubiquitination of IGPR-1. a A schematic of in vitro ubiquitination assay is shown. b In vitro ubiquitination assay was carried out as described in the material and method section using biotinylated ubiquitin (Ub), E2 conjugating enzymes, purified GST-NEDD4 and GST-cytoplasmic domain of IGPR-1. The samples were resolved on SDS-PAGE and ubiquitinated GST-IGPR-1 was detected by Western blotting via Streptavidin-HRP antibody. c–e Coomassie blue staining of GST-IGPR-1, GST-NEDD4 and E2 conjugating enzymes. f HEK-293 cells expressing IGPR-1-Myc were transfected with an empty vector or wild-type NEDD4-HA, cells were lysed and whole cell lysates were immunoprecipitated with an anti-Myc antibody followed by Western blot analysis using anti-ubiquitin (FK2) antibody, K63-poly-ubiqutin antibody or K48-poly-ubiqutin antibody. Whole cell lysates (WCL) were blotted for IGPR-1-Myc and NEDD4-HA