Fig. 4

NEED4 mediates K48- and K63-dependent polyubiquitination and downregulation of IGPR-1. a A schematic of ubiquitin (Ub) constructs are shown. b HEK-293 cells expressing IGPR-1 were transfected with wild-type Ub, KO-Ub, K48-Ub or K63-Ub constructs. After 48 h, cells were lysed and whole cell lysates (WCL) were immunoprecipitated with anti-Myc and blotted with anti-HA antibody for ubiquitin. The same membrane was re-blotted for IGPR-1. WCL was blotted for total IGPR-1, and loading control GAPDH. c IGPR-1/HEK-293 and IGPR-1/HEK-293 cells transfected with NEDD4 were subjected to cycloheximide pulse-chase assay. Cells were lysed at the indicated time points and whole cell lysates was subjected to Western blot analysis using anti-Myc antibody. The graph is representative of three independent experiments. Expression of NEDD4 and loading control GAPDH are shown. d HEK-293 cells expressing empty vector (EV), IGPR-1-Myc alone or co-expressing with NEDD4-HA were stained with anti-Myc antibody and subjected to immunofluorescence microscopy. Scale bar, 10 μm