FIGURE 8:

Knockout of Vps35 in 3T3-L1 adipocytes inhibits formation of small transport vesicles containing sortilin and Glut4 and blocks their delivery from peripheral endosomes to the perinuclear compartment. (A) Total lysates (L) prepared from wild-type and CRISPR/Cas9-edited 3T3-L1 adipocytes were fractionated into heavy membrane pellet (P1) and vesicular fractions (P2), which were analyzed by Western blotting (20 µg/lane). (B) Relative distribution of Glut4 and sortilin between fractions. Presence of each protein in total lysates (L) of wild-type and CRISPR/Cas9-edited 3T3-L1 adipocytes is 100%. **p < 0.01; *p < 0.05. Results were analyzed by Student’s t test. A representative result of three independent experiments. (C) Wild- type (top) and CRISPR/Cas9-edited (bottom) 3T3-L1 adipocytes were incubated with LysoTracker, fixed, and stained with rabbit polyclonal antibody against Glut4, followed by FITC-conjugated donkey anti-rabbit IgG, and analyzed by double immunofluorescence.