FIGURE 8.
Tumor-derived UBA mutations increase transforming activity of SCCRO. A, TCGA-identified mutations in the UBA domain augment the neddylation activity of SCCRO in vivo. A neddylation reaction on lysates derived from HeLa cells with and without pretreatment with MG132 shows that the levels of neddylated CUL1 and CUL3 are reduced by pretreatment with MG132 in MYC-SCCRO-transfected but not MYC-SCCROR13H- or MYC-SCCROQ20A-transfected cells. Results were confirmed by densitometry measurement. B, Western blot analysis of lysates from NIH 3T3 cells stably transfected with the indicated SCCRO constructs and probed with anti-MYC antibody showing equivalent expression. C, representative results from a soft agar assay showing that stable expression of TCGA-identified UBA domain mutants in NIH 3T3 cells results in increased colony formation in soft agar relative to expression of SCCRO. D, graph showing the average number of colonies for each NIH 3T3 clone from B. IB, immunoblotting.