Fig. 3: SYVN1 catalyses the ubiquitylation of MCT4. | Cell Death & Disease

Fig. 3: SYVN1 catalyses the ubiquitylation of MCT4.

From: SYVN1-mediated ubiquitylation directs localization of MCT4 in the plasma membrane to promote the progression of lung adenocarcinoma

Fig. 3

A, B In vivo ubiquitylation assay of MCT4 catalysed by SYVN1. Flag-tagged MCT4 was transfected into HEK293T cells along with HA-tagged SYVN1. Forty-eight hours post-transfection, cell lysates were immunoprecipitated with an anti-Flag antibody under denaturing conditions. The immunoprecipitates were analysed by immunoblotting with the indicated antibodies (A). B Amounts of ubiquitylated MCT4 in (A) were determined by densitometry of protein bands. Flag-MCT4 was used as the loading control. Data are presented as the mean ± SD. (**P < 0.01, n = 3, two-tailed unpaired t tests). C, D Reverse ubiquitylation assay of MCT4 catalysed by SYVN1. HEK293T cells were transfected with the indicated plasmids. Forty-eight hours post-transfection, the ubiquitylated proteins were purified under denaturing conditions via Ni-NTA agarose beads and analysed by immunoblotting with the indicated antibodies (C). D Amounts of ubiquitylated MCT4 in (C) were determined by densitometry of protein bands. PolyUb was used as the loading control. Data are presented as the mean ± SD. (**P < 0.01, n = 3, two-tailed unpaired t tests). E western blots show the knockdown of SYVN1. A549 and H1752 cells were infected with lentivirus carrying scramble control shRNA (scramble) or shRNAs targeting SYVN1. F In vivo ubiquitylation assay of endogenous MCT4 upon SYVN1 knockdown. A549 cells were transduced with scramble or SYVN1 shRNA lentiviral vector. Cell lysates were immunoprecipitated with anti-MCT4 antibody under denaturing conditions. The immunoprecipitates were resolved and analysed by western blotting. G The PLA assay demonstrates MCT4 ubiquitylation in cells with SYVN1 knockdown. Scale bar: 20 μm. H, I The quantification of the PLA signals measured in (G). Data are presented as the mean ± SD (**P < 0.05, n = 6, two-tailed unpaired t tests). J HEK293T cells were transfected with the indicated plasmid. Forty-eight hours post transfection, cell lysates were immunoprecipitated with an anti-Flag antibody under denaturing conditions. The immunoprecipitates were analysed by immunoblotting with the indicated antibodies. K Amounts of ubiquitylated MCT4 and its mutants in (I) were quantified by densitometry of protein bands. Flag-MCT4 was used as the loading control. Data are presented as the mean ± SD. (**P < 0.01, n = 3, two-tailed unpaired t tests). L HEK293T cells were transfected with the indicated plasmids. Forty-eight hours post transfection, the ubiquitylated proteins were purified under denaturing conditions via Ni-NTA agarose beads and analysed by immunoblotting with the indicated antibodies. M Amounts of ubiquitylated MCT4 and its mutants in (L) were quantified by densitometry of protein bands. PolyUb was used as the loading control. Data are presented as the mean ± SD. (**P < 0.01, n = 3, two-tailed unpaired t tests). N The PLA assay demonstrates the ubiquitylation of MCT4 and its deletion mutants upon SYVN1 overexpression. Scale bar: 20 μm. O Quantification of the PLA signals measured in (N). Data are presented as the mean ± SD (**P < 0.01, n = 6, two-tailed unpaired t tests).

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