Fig. 1: MCT4 is post-translationally modified by ubiquitylation in LUAD cells.

A MCT4 expression pattern was detected by western blotting. Smeared bands were observed. B MCT4 ubiquitylation was detected in an in vivo ubiquitylation assay. Flag-tagged MCT4 was transfected into HEK293T cells. Cell lysates were immunoprecipitated with anti-Flag antibody under denaturing conditions. The immunoprecipitates were resolved and analysed by western blotting. C The ubiquitylation of MCT4 was demonstrated by a reverse ubiquitylation assay. Flag-tagged MCT4 was transfected into HEK293T cells along with His-tagged ubiquitin. The ubiquitylated proteins were purified under denaturing conditions via Ni-NTA agarose beads and were resolved and analysed. D The ubiquitylation of MCT4 was visualized by an in-situ proximity ligation (PLA) assay with anti-MCT4 and anti-Ub antibodies. The PLA signal (red) represents the intensity of ubiquitylated MCT4. Blue, nucleus. Scale bar: 20 μm. E The PLA assay identifies the ubiquitylation of MCT4 in LUAD tissues. Scale bar: 50 μm. F Schematic diagram of the MCT4 molecule and its deletion mutants. G In vivo ubiquitylation assay using MCT4 deletion mutants. Flag-tagged MCT4 or its mutants were transfected into HEK293T cells. Cell lysates were subjected to denaturing immunoprecipitation and immunoblotted with the indicated antibodies. H Reverse ubiquitylation assay using MCT4 deletion mutants. Flag-tagged MCT4 or its mutants were transfected into HEK293T cells along with His-tagged ubiquitin. The ubiquitylated proteins were purified under denaturing conditions via Ni-NTA agarose beads and were resolved and analysed. I The ubiquitylation of Flag-MCT4 and its mutants in HEK293T cells was detected by PLA. Scale bar: 20 μm. J The quantification of the PLA signals measured in (I) was performed using ImageJ software. Data are presented as the mean ± SD (**P < 0.01, n = 5, two-tailed unpaired t tests).