Fig. 6: GMDS-AS1 blocks ubiquitination-dependent proteasomal degradation of HuR.

A The protein levels of HuR, p-STAT3 (Y705) and total STAT3 in HCT116 cells (left) and SW620 cells (right) stably transduced with control or GMDS-AS1 shRNA were detected by immunoblotting. β-actin was the endogenous control. B GMDS-AS1 control/KD HCT116 cells were treated with MG132 (50 μg/ml) or dimethyl sulfoxide (DMSO) for 8 h, and the protein level of HuR was detected by immunoblotting. Densitometric analyses were performed with ImageJ software. The relative level of HuR protein expression was calculated and is labeled below the charts (normalization on the basis of the β-actin expression level). C GMDS-AS1 control/KD HCT116 cells were treated with CHX (50 μg/ml) for the indicated times, and the protein expression level of HuR was detected by immunoblotting (left). Densitometric analysis curve of the HuR protein expression levels (right). β-actin was the endogenous control. D, E Immunoprecipitation (IP) experiments were performed to detect the polyubiquitination and K-48 polyubiquitination levels of HuR after GMDS-AS1 expression was knocked down in HCT116 cells. HCT116 cells stably transduced with control or GMDS-AS1 shRNA were transfected with hemagglutinin (HA)-ubiquitin (Ub)-expressing plasmids for 48 h. After treatment with 20 μM MG132 for 8 h, cell lysates were immunoprecipitated with either control IgG or an anti-HuR antibody and immunoblotted with an anti-ubiquitin antibody (D) and an anti-K48-Ub (E) antibody. HuR, Ub, K48-Ub and β-actin were the loading controls. F IP experiments were performed to detect an interaction between HuR and β-TrCP1 after GMDS-AS1 expression was knocked down in HCT116 cells. HCT116 cells stably transduced with control or GMDS-AS1 shRNA were transfected with MYC-β-TrCP-expressing plasmids for 48 h. The cell lysates were immunoprecipitated with control IgG and anti-HuR and anti-β-TrCP antibodies and immunoblotted with anti-HuR and anti-β-TrCP antibodies. HuR, β-TrCP and β-actin were the loading controls.