Figure 3

Cops2 binds to and stabilize Nanog protein.

(a) Three days after transfection, GFP, Cops2, Cops5 and Cops8 KD cells were treated with cycloheximide for indicated time. Nanog protein levels were measured by Western blot. (b) Quantification results of (a). Data are shown as mean ± SD (n = 3). (c) Schematic illustration of Nanog protein. ND: N-terminal domain; HD: Homeobox domain; CD: C-terminal domain. Four lysine residues in the α3 of the HD are shown in green. (d) Cops2 was knocked down in ESCs expressing Flag-tagged ΔND, ΔHD, or ΔCD Nanog mutants. The expression levels of endogenous Nanog and exogenous mutants were measured by Western blot. ΔND Nanog mutant was detected by Flag antibody. (e) Similar to (d), except that ESCs expressing Flag-tagged Δα1, Δα2, or Δα3 Nanog mutants were used. (f) HEK293T cells were transfected with plasmids expressing Cops2 or Nanog, as indicated. Two days after transfection, Co-IP and Western blot were performed. (g) Co-IP experiments to detect the interactions of Cops2 with Flag-tagged WT, Δα1, Δα2 and Δα3 Nanog. (h) Three days after transfection, GFP and Cops2 KD ESCs were treated with 100 nM bafilomycin A1 (BA), 10 μM MG132, or 60 ng/ml leptomycin B (LMB) for 2 hours and then subjected to Western blot. (i) Cops2 was knocked down in ESCs expressing 4KR ΔCD Nanog mutant, in which four lysine residues (K138, K141, K150 and K152) in α3 were replaced by arginine. Western blot was performed to measure the expression of endogenous Nanog and exogenous 4KR ΔCD Nanog.