Fig. 4

Low level of RNF128 regulates melanoma cellular EMT and stemness via simultaneously protecting CD44 and CTTN from degradation. a Identification of the binding partners of RNF128 by a combination of Co-IP and MS. b Venn diagram showing the number of binding partners of RNF128 in A2058-RNF128, M14-RNF128, and HEK-293 T-RNF128 (left), and 10 overlapping proteins are presented in the table (right). c Co-IP (left) and immunofluorescence assays (right) were used to detect the direct interaction of RNF128 and CD44. d Western blot and qRT-PCR were used to detect the expression of RNF128 and CD44 in the indicated cells. Low level of RNF128 upregulated the expression of CD44 both in protein and mRNA level, while interfered CD44 did not influence the RNF128 expression. e Co-IP (left) and immunofluorescence assays (right) were used to detect the direct interaction of RNF128 and CD44. f Western blot and qRT-PCR were used to detect the expression of RNF128 and CTTN in the indicated cells. Low level of RNF128 upregulated the expression of CTTN in protein level, while interfered CTTN did not influence the RNF128 expression. g Impact of M14-shNC and M14-shRNF128 cells on CD44 or CTTN ubiquitination. V5-CD44 or V5-CTTN and HA-Ubi were co-transfected into M14-shNC and M14-shRNF128 cells, and the cell lysates were immunoprecipitated with the indicated antibodies and analyzed by western blot. h The degradation of CD44 and CTTN was weakened in M14-shRNA128 cells compared with that in M14-shNC cells, as demonstrated by CHX chase assays. Cells were exposed to CHX and collected at the indicated times. Cell lysates were immunoblotted with the indicated antibodies. i Invasion and colony formation assays were performed in the indicated cells (left); western blot was used to detect the expression of RNF128, CD44, CTTN, EMT markers (E-cadherin, vimentin, and Snail), and stemness markers (CD133) in the indicated cells (right). j Invasion and colony formation assays were performed in the indicated cells (left); western blot was used to detect the expression levels of RNF128, CD44, CTTN, EMT markers (E-cadherin, vimentin, and Snail), and stemness markers (CD133) in the indicated cells (right). n.s., not significant, ^**p < 0.01, ^***p < 0.001, ^****p < 0.001