Fig. 5
From: USP8 maintains embryonic stem cell stemness via deubiquitination of EPG5
USP8 deubiquitinates the EPG5. a Western blot analysis of whole-cell extracts from WT, Usp8-overexpression and Usp8▵/− ESC lines using anti-EPG5 antibody. β-Actin served as a loading control. Images are representative of three independent experiments. b Lysates from WT, Usp8-overexpression and Usp8▵/− ESC lines were immunoprecipitated with anti-EPG5 and immunoblotted with anti-ubiquitin. Input cell lysates were immunoblotted with anti-USP8 and β-actin as controls. c Lysates of HEK293T cells transfected with plasmids encoding Flag-Epg5, HA-Ubiquitin and Myc-Usp8 were immunoprecipitated with anti-Flag and immunoblotted with anti-HA and anti-FLAG. Input cell lysates were immunoblotted with anti-Myc, anti-HA, and anti-GAPDH as controls. d Lysates of HEK293T cells transfected with plasmids encoding Flag-Epg5, HA-Ubiquitin, and Myc-Usp8 were immunoprecipitated with anti-Flag and immunoblotted with anti-K63, anti-K48, and anti-HA antibodies. e Lysates of HEK293T cells transfected with plasmids encoding Flag-Epg5, HA-Ubiquitin, or HA-Ubiquitin (K63R) were immunoprecipitated with anti-Flag and immunoblotted with anti-HA and anti-FLAG antibodies. Input cell lysates were immunoblotted with anti-FLAG, anti-HA and anti-GAPDH antibodies as controls. f Proteins from ESCs expressing Flag-Epg5 were analyzed by LC-MS/MS mass spectrometry. Ubiquitination sites identified on EPG5 are indicated. g)Immunoblot analysis of protein extracts of HEK293T cells transfected with HA-Ubiquitin together with plasmids expressing WT Flag-Epg5 or K200R, K252R, K1115R, K1265R, K1615R, and K1948R mutants of Flag-Epg5
