Research Paper Volume 12, Issue 3 pp 2373—2392

Figure 4. UBE2M regulates the expression of cyclin D1 through stabilizing β-catenin. (A) The protein expression level of β-catenin, cyclin D1, and c-Myc in UBE2M-silencing PLC/PRF/5 and MHCC-97L cells, as well as in UBE2M-overexpressing SMMC-7721 and BEL-7402 cells by Western blotting. (B) MHCC-97L cells transfected with NC siRNA and si-UBE2M for 48h, prior to addition of CHX (100 μg/mL). Cell lysates were prepared at the indicated times (0, 1, 2, 3, and 4 h) following addition of CHX, and analyzed by Western blotting. (C) Quantification of β-catenin protein levels in MHCC-97L cells at the indicated times as described in (B). (D) Nuclear and cytoplasmic fractions of MHCC-97L and BEL-7402 cells were isolated, and β-catenin expression was determined in the nuclear and cytoplasmic protein by Western blotting. (E) Nuclear and cytoplasmic expressions of UBE2M and β-catenin were analyzed by immunofluorescence staining in the MHCC-97L and BEL-7402 cells (Scale bar, 15 μm). (F) The physical interaction between UBE2M and β-catenin was examined by a co-IP assay in MHCC-97L cells. IgG was used as a negative control.