Fig. 3. Stomatin interacted with CD36 and facilitated fatty acid uptake.
a Immunoprecipitation (IP) assays revealed molecular interactions between stomatin and CD 36 in vitro. b Fatty acid uptake assays were performed to measure internalization of lipid ingredients by adipocyte-like cells from the extracellular environment. Fluorescently-labeled fatty acid (Bodipy-FL-C16) or fluorescent tag alone (Bodipy-FL) was added to the culture medium of adipocyte-like cells expressing exogenous stomatin (hSTOM), or control vector (Ctl). The degrees of Bodipy-FL-C16 and Bodipy-FL internalization were measured by intracellular accumulations of fluorescence over time. Mean ± s.e.m. for at n = 3 for Bodipy-FL-C16 n = 6 for Bodipy-FL uptake independent cells. c Immunofluorescence staining demonstrated that in cells treated with BSA, about half of the cell population contained stomatin-associated LDs. Treating cells with palmitic acid (PA) resulted in dissociation of STOM from LDs. Mean ± s.d. for three independent experiments. *P < 0.05 by two-sided unpaired t-test. Bar = 10 µm. d Surface biotinylation followed by Western blotting analyses of the biotinylated proteins were performed to quantify the surface portion (Surface) of a membrane protein, including stomatin, CD36, caveolin-1 (CAV1), or ATP1A1, relative to the total amount of individual proteins (Input). Actin proteins could not be labeled by surface biotinylation method and therefore contained no surface portion. Note increase of surface portion of stomatin after PA treatments (arrow) compared to the BSA control. In contrast, the surface portion of CD36, CAV1 remained unchanged after PA treatments. Mean ± s.d. for three independent experiments. *P < 0.05 by two-way ANOVA analysis. e, f Proximity ligation assays (PLA) were performed in 3T3-L1 adipocyte-like cells over-expressing exogenous human stomatin fused with GFP (hSTOM-GFP). Duolink® PLA reactions occurred when PLA probes for CD36 e or for CAV1 f were in closed proximity with hSTOM-GFP. Addition of connector oligos joined the PLA probes, resulting in in situ proximity ligation and formation of amplicon (arrowheads). e PA treatments promoted interactions between CD36 and stomatin on the plasma membrane, as evidence by the increased amplicons along cell surface compared with the control BSA treatments; removal of PA reversed such an increase. On the other hand, f PA treatments did not affect interactions between calveolin-1 and stomatin. Bar = 10 µm. Source data are provided as a Source data file.