Fig. 6

TMEM120B stabilized MYH9 by preventing its ubiquitin-mediated degradation from CUL9. (A and B) Western blotting and qPCR assays were performed to determine the protein and mRNA expression of MYH9, respectively, after overexpressing or deleting TMEM120B in SK-BR-3 or MDA-231 cells. (C-D) Western blotting and qPCR assays were performed to assess the protein and mRNA expression of TMEM120B, respectively, after overexpressing or deleting MYH9 in SK-BR-3 or MDA-231 cells. (E) After being treated with CHX at indicated time points, the expression of MYH9 was evaluated by western blotting after overexpressing or silencing TMEM120B in SK-BR-3 or MDA-231 cells. (F) The ubiquitination level of MYH9 was detected using western blotting after being transfected with TMEM120B, TMEM120B-∆CCD, and control plasmids in SK-BR-3 cells. (G) Mass spectrometry (MS) analysis was performed to identify candidates for interaction with ectopic TMEM120B or TMEM120B-∆CCD in SK-BR-3 cells, respectively. (H) Endogenous co-IP assay was performed to detect the interaction between MYH9, CUL9, and TMEM120B in MDA-231 cells. (I and J) Protein levels of MYH9 and the ubiquitination level were assessed using western blotting after transfecting TMEM120B alone or co-transfecting both TMEM120B and CUL9 in SK-BR-3 cells. (K) Co-IP assay was used to evaluate the interaction among TMEM120B, MYH9, and CUL9 after overexpressing TMEM120B and CUL9 in different doses in SK-BR-3 cells. Quantification data are expressed as mean ± SD of three independent experiments (t-test, two-sided)