Fig. 6

RING-domain mutant CGRRF1 (C274A/C277A) interacts with EGFR but does not ubiquitinate EGFR. a, b Immunofluorescence analysis (×100) shows colocalization of FLAG-tagged CGRRF1 (green) and Myc-tagged EGFR (red) in both starvation and EGF stimulation conditions. White blank boxes (right columns) show high magnification of CGRRF1 and EGFR colocalization from merge columns. U2OS cells were transiently co-transfected with FLAG-tagged wild-type CGRRF1 (a) or mutant CGRRF1 (b) with Myc-tagged EGFR for 24 h. Transfected cells were starved in serum-free, 0.1% BSA-containing medium for 24 h and then stimulated with EGF (50 ng/ml). c MDA-MB-231 cells were transfected with FLAG-tagged wild-type or mutant CGRRF1. Cells were harvested 48 h after transfection, and lysates were pulled down with anti-FLAG beads. d CGRRF1 doxycycline-inducible MDA-MB-231 cells were transfected with pcDNA3 or HA-UbK48. The next day after transfection, cells were serum-starved and treated with 100 ng/ml doxycycline. Twenty-four hours after starvation, cells were stimulated with 100 ng/ml EGF for 5 min. Lysates were then harvested for in vivo ubiquitination assay. Induced CGRRF1 proteins were indicated by arrows. The bar graph represents data from four independent experiments. Error bars represent mean ± SD. *p < 0.05