Fig. 5

Highly expressed p-ANXA2 (Tyr23) inhibits the ubiquitin-dependent degradation of MYC protein in ESCC cells. a ESCC cells were co-transfected with pcDNA3.1-ANXA2-Y23D and pcDNA3.1-MYC, and localization of ANXA2-Y23D (green) and MYC (red) was detected by immunofluorescence staining. Scale bar = 30 μM. b ESCC cells stably expressing shANXA2 were transfected with pcDNA3.1-ANXA2-Y23A, pcDNA3.1-ANXA2-Y23D, or empty vector. The indicated proteins were analyzed by western blotting. GAPDH was used as the loading control. An anti-His antibody was used to detect the exogenously expressed ANXA2 protein. c Nucleoprotein was extracted from ESCC tumor (T) and adjacent normal (N) tissues, and MYC and p-ANXA2 protein levels were detected by western blotting. Histone H3 was used as the loading control for nuclear protein. d ESCC cells transfected with either a pcDNA3.1-ANXA2-Y23A or pcDNA3.1-ANXA2-Y23D construct were treated with the indicated drugs and subjected to a Western blotting analysis. e Cells were treated with cycloheximide (CHX) for different lengths of time and MYC protein levels were examined by Western blotting. GAPDH was used as the loading control. B, Cells were treated with the proteasomal inhibitor MG132 and subjected to an immunoprecipitation assay with MYC antibody. The level of ubiquitinated MYC was detected via western blotting with a ubiquitin antibody