Fig. 4: Ubiquitination of MCT4 determines its localization in the plasma membrane.

A, B SYVN1 does not affect the protein stability of MCT4. A549 and H1752 cells infected with lentiviruses carrying the indicated shRNAs were treated with CHX (500 μg/ml) and harvested at the indicated time followed by western blotting analysis. C MG132 treatment does not alter MCT4 abundance. A549 and H1752 cells infected with lentiviruses carrying the indicated shRNAs were treated with MG132 (10 μM) for 8 h. D, E HEK293T cells were transfected with the indicated plasmids. Forty-eight hours post transfection, cell lysates were immunoprecipitated with anti-Flag antibody (D) or pulled down by Ni-NTA agarose beads (E) and then immunoblotted with the indicated antibodies. F HEK293T cells were transfected with the indicated plasmids. The interaction between MCT4 and CD147 was detected by a PLA assay with anti-Flag and anti-His antibodies. The PLA signal represents the intensity of the interaction. Scale bar: 20 μm. G Quantification of the PLA signals measured in (F) was performed using ImageJ software. Data are presented as the mean ± SD. (n.s., no significance, n = 4, two-tailed unpaired t tests). H The interaction between MCT4 and CD147 did not obviously change based on the PLA assay. Blue, nucleus. Scale bar: 20 μm. I The subcellular localization of MCT4 (green) in cells infected with lentivirus carrying scramble control shRNA (scramble) or shRNAs targeting SYVN1 was determined by an immunofluorescence assay. Blue, nucleus. Scale bar: 20 μm. J A549 and H1752 cells were infected with lentivirus carrying scramble control shRNA (scramble) or shRNAs targeting SYVN1. Total cell lysate (T), cytosol fraction C and plasma membrane fraction (PM) were analysed by immunoblotting with the indicated antibodies. K, L Quantification of MCT4 signals in different cellular fractions. (*P < 0.05, **P < 0.01, n = 3, two-tailed unpaired t test).