Figure 2: Plin1 interacts with Fsp27.
From: Perilipin1 promotes unilocular lipid droplet formation through the activation of Fsp27 in adipocytes
(a) Fsp27 and Plin1 interact with each other in 3T3-L1 adipocytes. Anti-Fsp27 antibodies were used to immunoprecipitate Fsp27 (IP) from 8-day differentiated 3T3-L1 adipocytes lysates and the presence of Plin1 or ADRP was detected by antibodies against Plin1 or ADRP. Pre-immune serum was used as a negative control. H, heavy chain. (b) Similar to a, except that Plin1 or ADRP/Plin2 were immuneprecipitated (IP) by anti-Plin1 or anti-ADRP antibodies. The presence of Fsp27 in the immunoprecipitated products was detected by anti-Fsp27 antibodies. (c) Acidic motif (a.a. 291–318) of Plin1 mediates its interaction with Fsp27. Full-length Flag-Fsp27 was co-transfected with full-length (FL) or indicated truncations of HA-Plin1 into 293T cells. Flag-Fsp27 was immunoprecipitated by anti-FLAG M2-agarose beads. IB, immunoblotting; H, heavy chain; L, light chain. Red asterisks mark the positions of Plin1 truncations. Co-immunoprecipitation experiments were performed similarly in d, f and g. (d) Deletion of the acidic motif (AM) in the mid-region of Plin1 (Plin1ΔAM) abolishes the Fsp27–Plin1 interaction. (e) Schematic diagram of Plin1 truncations. Right panel summarizes the results from c and d. (f) The CIDE-N domain (a.a. 38–120) of Fsp27 mediates its interaction with Plin1. Red asterisks mark the HA-Plin1 co-precipitated with indicated Fsp27 truncations. Red arrow points to Fsp27 (a.a. 38–217), which overlaps with the light chain (L). (g) Flag-tagged Fsp27, but not Cidea or Cideb, interacts with Plin1(233–405). (h) Representative images showing that Plin1 can enhance Fsp27’s activity in promoting large LD formation through their interaction. Wild-type Fsp27 (pCDNA-WT-Fsp27) was co-expressed with HA-tagged Plin1 or Plin1(233–405) or Plin1ΔAM in 3T3-L1 preadipocytes. Fsp27 proteins are stained with antibody against Fsp27 (red). LDs are stained with BODIPY 493/503 (green). Inserted boxes are enlarged area in the relevant images. Scale bars, 5 μm. (i) Plin1 enhances Fsp27’s activity and the expression of Plin1 alone cannot promote LD growth. Large LDs are defined as LDs with a diameter ≥2.5 μm. The diameter of LDs was measured in total 1,000–1,500 cells from three independent experiments (mean±s.d., one-way ANOVA Tukey test, **P<0.01; NS, no significant difference).
