Fig. 5

CGRRF1 ubiquitinates EGFR through K48-liked ubiquitination. a CGRRF1-knockdown BT-549 cell lines were transfected with pcDNA3 or HA-UbK48. Lysates were harvested 48 h after transfection and then subjected to in vivo ubiquitination assay as described in “Methods.” b CGRRF1-overexpressing BT-549 cell lines were transfected with pcDNA3 or HA-UbK48. Lysates were harvested 48 h after transfection and subjected to in vivo ubiquitination assay. c CGRRF1-knockdown MDA-MB-231 cell lines were transfected with pcDNA3 or HA-UbK48. The next day, cells were serum-starved for 24 h and then treated with 100 ng/ml EGF for 30 min. Cell lysates were harvested and in vivo ubiquitination assay was performed. The CGRRF1 signal was indicated by an arrow. d CGRRF1-overexpressing BT-549 cell lines were transfected with pcDNA3 or HA-UbK48. The next day, cells were serum-starved for 24 h and then treated with 100 ng/ml EGF for 5 min. Cell lysates were then harvested for in vivo ubiquitination assay