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. 2022 Sep 16;60(4):359–367. doi: 10.1136/jmg-2022-108602

Figure 4.

Figure 4

VPS35L deficiency impairs cell surface low-density lipoprotein receptor (LDLR) level and low-density lipoprotein (LDL) uptake. (A) Representative immunoblots of 3T3 parental, VPS35L-knockout (KO) and sh-VPS35L-knockdown cells. glyceraldehyde-3-phosphate dehydrogenase(GAPDH) and transferrin receptor(TFNR) immunoreactivity indicate equivalent loading. (B) Protein intensity of LRP1, LDLR and TFNR relative to parental cells averaged over three independent experiments. (C) HEK293T cells were transfected with either HA-VPS35L alone or HA-VPS35L and FLAG-LDLR. Interaction between HA-VPS35L and FLAG-LDLR was detected via immunoprecipitation with anti-HA antibody. (D) HEK293T cells were transfected with FLAG-LDLR, and interaction with endogenous VPS35L was detected by immunoprecipitation with anti-FLAG antibody. (E) Representative images of DiI-LDL uptake in parental, VPS35L-KO and sh-VPS35L 3T3 cells. Cells were incubated with DiI-LDL for 30 min, followed by DAPI staining and imaging with a fluorescence microscope. (F) DiI intensity was quantified using ImageJ software. We acquired 30 fields percondition, and DiI intensity of each field was normalised to the number of DAPI nuclei. Bar graphs (B) and (F), means and SEM are shown, **p<0.01.