Fig. 5

MAP4 is a novel target of FBXW7 via the phosphorylated threonine T521 modified by CHEK1 in ESCC. A Image of SDS-PAGE gel includes protein complex that is GST-FBXW7 incubated with cell lysate or PBS. B Schematic diagram of the typical FBXW7 recognition motif, the potential protein sequence in MAP4 and CHEK1 phosphorylation motif. C Immunoprecipitation analysis of the interaction between FBXW7 and MAP4 in KYSE70 cells. D Western blot analysis of MAP4, FBXW7 and GAPDH in negative control and FBXW7 knockout KYSE70 and KYSE180 cells. E FBXW7-KO cells (gFBXW7-1) transfected with FBXW7 plasmid or EV (empty vector) were treated with cycloheximide (CHX) for different lengths of time and MAP4 protein levels were examined by Western blot. GAPDH was used as the loading control. F FBXW7-KO cells transfected with FBXW7 plasmid or EV were treated with DMSO or the proteasomal inhibitor MG132 and MAP4 protein levels were examined by Western blot. GAPDH was used as the loading control. G FBXW7-KO cells transfected with FBXW7 plasmid or EV were treated with the proteasomal inhibitor MG132 and subjected to an immunoprecipitation assay with MAP4 antibody. The level of ubiquitinated MAP4 was detected via Western blot with a ubiquitin antibody. H Western blot analysis of MAP4 and GAPDH expression in KYSE70 and KYSE180 cells transfected with different MAP4 plasmids. I Immunoprecipitation analysis of the interaction between FBXW7 and different MAP4 plasmids in KYSE70 cells. J Western blot analysis of p-ERK, ERK, VEGFA, MMP3, FBXW7, MAP4 and GAPDH in the negative control, CHEK1-overexpressing KYSE70 and KYSE180 cells treated with the CHEK1 inhibitor CHIR-124. K Western blot analysis of MAP4, p-ERK, ERK, MMP3, VEGFA, FBXW7 and GAPDH in negative control and MAP4 knockdown KYSE70 and KYSE180 cells