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. 2017 Jun 15;28(12):1667–1675. doi: 10.1091/mbc.E16-11-0777

FIGURE 4:

FIGURE 4:

Sortilin interacts with the first luminal loop of Glut4 and retromer. (A) 3T3-L1 adipocytes stably transfected with myc7-Glut4 were stained with mouse monoclonal anti-myc antibody and rabbit polyclonal sortilin antibody, followed by Texas red–conjugated donkey anti-mouse IgG and FITC-conjugated donkey anti-rabbit IgG and analyzed by double immunofluorescence. (B) Total lysates (0.5 mg) prepared from wild-type (WT) 3T3-L1 adipocytes and 3T3-L1 adipocytes stably transfected with sortilin-myc/His (S) were incubated with HisPur cobalt resin; beads were washed and additionally incubated with the peptide (100 ng) corresponding to the first luminal loop of Glut4. Eluates were analyzed by electrophoresis in Tricine gels and Western blotting. A representative result of three independent experiments. (C) The cytoplasmic tail of sortilin (CT) in the GST vector along with GST alone as a control were prebound to glutathione beads and then incubated with the cytoplasmic extract (0.5 mg) from insulin-treated and untreated 3T3-L1 adipocytes. Eluates along with input and unbound material (equal volume aliquots) were analyzed by SDS electrophoresis and Western blotting. A representative result of three independent experiments. (D) 3T3-L1 adipocytes stably expressing myc7-Glut4 were stained with mouse monoclonal anti-myc antibody, goat polyclonal antibody against Vps35, and rabbit polyclonal antibody against sortilin, followed by Alexa Fluor 647–conjugated donkey anti-mouse IgG, Alexa Fluor 546–conjugated donkey anti-goat IgG, and FITC-conjugated donkey anti-rabbit IgG and analyzed by immunofluorescence. (E) 3T3-L1 adipocytes stably expressing myc7-Glut4 or myc7-Glut4 along with sortilin-myc/His were cross-linked with DSP; sortilin-myc/His-containing complexes were isolated on HisPur cobalt resin as described in Materials and Methods and analyzed by Western blotting. A representative result of three independent experiments. (F) Sortilin may work as a transmembrane scaffold linking Glut4 to the retromer-mediated protein traffic.