Fig. 2: RNF180 promotes the degradation of RhoC protein by increasing its ubiquitination level and interacts with RhoC.

A Increasing concentrations of RNF180 plasmid were transfected into HEK293T cells for 36 h and the protein level RhoC were analyzed by immunoblotting. B The mRNA level of RNF180 and RhoC were analyzed by RT-qPCR assay. C HEK293T cells were co-transfected by RhoC plasmid together with either RNF180 plasmid or control vector. After 36 h, cells were treated with 100 μg/ml CHX at the indicated time point. The cell lysates were subjected to immunoblotting and RhoC expression was quantified by ImageJ software. D The RNF180 plasmid was co-transfected transiently with RhoC-FLAG plasmid or control vector into HEK293T cells and 36 h later, cells were incubated with 5 μM MG132 for 6 h. Cell lysates were immunoprecipitated to pull down RhoC by the FLAG M2 affinity gel and subjected to immunoblotting. 5% of cell lysates were used to examine the total expression of RhoC and RNF180. E RNF180-FLAG plasmid or control vector was co-transfected transiently with the RhoC plasmid into HEK293T cells and the co-immunoprecipitation assay was performed as described in D. F HA-Ub and RhoC-FLAG plasmids were co-transfected with RNF180 in HEK293T cells. After 36 h, cells were treated with 5 μM MG132 for 6 h. RhoC-FLAG protein was immunoprecipitated and analyzed by immunoblotting. The poly-ubiquitination level of RhoC was detected by the anti-HA antibody.