Research Paper Volume 12, Issue 11 pp 10275—10289

Figure 5. Ubiquitin-conjugating enzyme E2T (UBE2T) maintains GRP78 stability and regulates epithelial-mesenchymal transition (EMT) markers. (A) UBE2T was depleted in LN229 and U251 cells. Cell lysates were examined using the indicated antibodies. (B) Immunoblotting of UBE2T, GRP78 and actin in LN229 and U251 cells transduced with UBE2T short hairpin RNA (shRNA) in the absence or presence of 10 μmol/L MG132 for 8 h. (C) U251 cells transfected with the control shRNA vector (ShVector) were treated with cycloheximide (100 μg/mL) and collected at the indicated times for immunoblotting. Quantification of GRP78 expression relative to β-actin expression is shown. (D) U251 cells transfected with ShUBE2T-1 were treated with cycloheximide (100 μg/mL) and collected at the indicated times for immunoblotting. Quantification of GRP78 expression relative to β-actin expression is shown. (E) Immunoblotting of UBE2T, GRP78, actin and EMT biomarkers (E-cadherin, N-cadherin and vimentin) in LN229 and U251 cells transfected with ShUBE2T-1, ShUBE2T-2 or ShVector. (F) Immunoblotting of UBE2T, GRP78, N-cadherin, E-cadherin, vimentin and Actin in LN229 and U251 cells transduced with the indicated plasmids. (Error bars indicate the SEM of three independent experiments).