Identification of VPS13C as a Galectin-12-Binding Protein That Regulates Galectin-12 Protein Stability and Adipogenesis (original) (raw)
Fig 1
Identification of VPS13C as a galectin-12-binding protein.
(A) Lysates from 3T3-L1 adipocytes transduced with a control retrovirus (Ctr) or one that expresses 3xFLAG tagged mouse galectin-12 (3Fm12) were immunoprecipitated with either an anti-FLAG or anti-galectin-12 antibody, as indicated. Immunoprecipitates were further immunoblotted with a galectin-12 antibody. (B) Immunofluorescence of 3T3-L1 adipocyte expressing 3Fm12 with anti-FLAG antibody (red). Cells were co-stained with Bodipy 493/503 and Hoechst 33342 to reveal lipid droplets (LD, green) and the nucleus (blue), respectively. Scale bar, 10 μm. (C) Lysates of control 3T3-L1 adipocytes or those expressing 3Fm12 were immunoprecipitated with anti-FLAG antibody and the immunocomplexes were analyzed by LC-MS/MS. (D) Lysates from 293T cells transducedwith 3xFLAG-tagged LC3 (control), full-length galectin-12 or truncated mutants lacking the N- or C-terminal CRD were immunoprecipitated with anti-FLAG M2-agarose beads, and total lysates or immunoprecipitates were analyzed by immunoblotting with anti-VPS13C or anti-FLAG antibodies. H and L denote the heavy and light chains of IgG, respectively. (E) Cytosol and lipid droplet fractions of 3T3-L1 adipocytes were immunoblotted with indicated antibodies. Results are representative of three experiments.