FIGURE 6.

CD166 enhanced YAP protein stability. A, CD166 decreased YAP degradation by actinomycin D. YAP was detected in empty plasmid vector or CD166-expressing plasmid-transfected Bel-7402 cells with or without (DMSO) actinomycin D (10 μg/ml) treatment. B, relative YAP expression was detected by qPCR (top) and Western blotting (bottom) under the same conditions as in A. Protein levels were regarded as the ratio of YAP/GAPDH. C, CD166 reduced YAP ubiquitination. Bel-7402 cells transfected with expressing constructs as indicated were treated with MG132 (25 μm) for 5 h before harvest. Exogenous YAP-FLAG was immunoprecipitated, and Western blotting analysis was done using anti-FLAG or anti-HA antibody. S, shorter exposure; L, longer exposure. D and E, CD166 protected YAP from degradation. Protein synthesis was blocked by treatment of cycloheximide (CHX; 50 μg/ml) for the indicated time in control (infected with GFP shRNA) and Bel-7402 cells with CD166 knocked down (infected with CD166-sh1) (D). Relative YAP protein levels were quantified by YAP/GAPDH ratio (E). Error bars, S.D.