Fig. 7: SLC35D3 increased the ER accumulation of NOTCH1 by interacting with the NOTCH1 extracellular domain.

a, b CoIP assay results showed the interaction between SLC35D3 and NOTCH1 (a), as well as the interaction between SLC35D3 and ECD (b). Representative images from three independent experiments are shown. c Direct interaction between SLC35D3 and ECD was further confirmed by pull-down assay. Representative images from three independent experiments are shown. d, e SLC35D3 overexpression promoted the colocalization of NOTCH1 and ER. Representative confocal images of cells are shown (d), as well as the quantification results of four independent parallel experiments (e). The inset in the right panel shows a magnified view of the indicated area. Scale bar, 20 μm. CALNEXIN, an ER marker protein. f, g SAKO mice showed higher levels of Notch1 at the cell membrane in IngWAT than control mice. Representative confocal images are shown (f), as well as the quantification results of six independent parallel experiments (g). Scale bar, 50 μm. Na-K ATPase, a plasma membrane marker protein. h–j Membrane proteins and endoplasmic reticulum proteins of IngWAT were isolated from SAKO and SAKI mice (n = 3 per group). The western blot results are shown (h), as well as the quantitative analysis results (i, j). Total NOTCH1 expression was normalized to β-Actin expression, membrane NOTCH1 expression was normalized to Na-K ATPase, and ER NOTCH1 expression was normalized to CALNEXIN. k–m Primary adipocytes were isolated from the IngWAT of SAKO mice and SAKI mice (n = 3 per group). Cell surface biotinylation assay results are shown (k), as well as the quantitative analysis results (l, m). Data are expressed as the mean ± SEM. Statistical significance was assessed by unpaired two-sided Student’s t test (e, g, i, j, l, m). The exact P values are shown in the figure. SAKO Slc35d3 adipocyte-specific knockout, SAKI Slc35d3 adipocyte-specific knock-in, OE overexpression, IngWAT inguinal white adipose tissue, ER endoplasmic reticulum. Source data are provided as a Source Data file.