Figure 5. SnoN sumoylation contributes to the ability of SnoN to antagonize TGFβ-suppression of E-cadherin.

A) Wild type SnoN and the SUMO loss of function SnoN (SnoN (KdR)) are expressed at equivalent levels in NMuMG stable cells. Lysates of NMuMG cells stably transfected with the control vector expressing the resistance marker alone or together with wild type SnoN (WT) or SUMO loss of function SnoN (KdR) were subjected to FLAG immunoprecipitation followed by SnoN immunoblotting (upper panel), or were immunoblotted for SnoN and actin, the latter serving as a loading control (lower two panels). B and C) Wild type but not the SUMO loss of function SnoN inhibits TGFβ-reduction in E-cadherin protein level in cells undergoing EMT. B) Representative scans of E-cadherin and actin immunoblots of lysates of control vector, wild type SnoN (WT) or SUMO loss of function SnoN (KdR) expressing cells that were left untreated or incubated with TGFβ. C) The densities of E-cadherin and actin immunoblots as shown in Figure 5B were analyzed (see MATERIALS and METHODS). Each column in the bar graph represents the mean (± SEM, n = 4) of percent suppression of E-cadherin protein level by TGFβ. D) Representative micrographs of E-cadherin-indirect immunofluorescence of vector control (−), wild type (WT) or SUMO loss of function (KdR) SnoN expressing NMuMG cells that were left untreated (I) or incubated with 20 pM (II) or 100 pM (III) TGFβ for 48 h. E) Intensity of E-cadherin immunofluorescence per cell was obtained and analyzed (see MATERIALS and METHODS). Each column in the bar graph represents the mean (± SEM, n = 3) of percent reduction in E-cadherin levels by 20 pM (clear column) or 100 pM (grey column) TGFβ. * indicates statistical significant difference (p<0.05) as compared to the vector control.