Fig. 6
CASZ1 decreases RAF1 expression by reducing the protein stability of RAF1 a. HCCLM3 cells transfected with CASZ1 were fixed for the double immunofluorescence staining, and colocalization of CASZ1 with RAF1 were visualized as yellow fluorescence in merge panel. Scale bar, 25 μm. b Co-immunoprecipitation assay was performed to analyze the direct binding between CASZ1 and RAF1 in HCCLM3CASZ1, HCCLM3 and PLC/PRF/5 cells. c The expression of RAF1 was determined in CASZ1-interfered HCC cells by western blot. d Correlation between CASZ1 and RAF1 was measured in 50 HCC samples. Representative IHC staining of CASZ1 and RAF1 (left panel, magnification, × 400) in serial sections are shown, indicating a negative correlation between the protein level of CASZ1 and RAF1 in the clinical samples (r = − 0.643, P < 0.001, right panel). e The half-life of RAF1 protein in HCC cells was analyzed following treatment with cycloheximide (CHX, 25 μg/ml) for the indicated time points. The half-life of RAF1 protein was decreased in HCCLM3 cells with CASZ1 overexpression, but increased in PLC/PRF/5 cells with CASZ1 knockdown. f MG-132 (20 μM) was used to inhibit the proteasomal degradation in HCCLM3 cells. MG-132 treatment reversed the downregulation of RAF1 protein induced by CASZ1 overexpression. *** P < 0.001