FIGURE 6.

Fsp27 is also localized to the ER, and increased Fsp27 following β-agonist treatment mainly associates with LDs. A, Fsp27 is present in the ER-enriched fraction in differentiated 3T3-L1 adipocytes. A biochemical fractionation experiment was performed using differentiated 3T3-L1 adipocytes (8 days after induction). The same percentage of each sample was loaded onto a SDS-polyacrylamide gel. 3T3-L1 preadipocytes were used as a negative control for Fsp27 expression. B, Fsp27 is co-localized with the ER-specific protein CB5 in 3T3-L1 adipocytes. 3T3-L1 adipocytes (7 days after induction) were electroporated with an expression plasmid encoding GFP-CB5. Endogenous Fsp27 was stained using a rabbit anti-Fsp27 antibody (red). GFP-CB5 (green) was used as an ER marker to visualize the ER network. Scale bar, 5 μm. C, increased Fsp27 protein level in the LD fraction of Iso (2 h)-treated 3T3-L1 adipocytes. HDM, high density microsome; LDM, low density microsome; Cyt, cytosol. A similar percentage of each fraction was loaded. D and E, increased Fsp27 protein level in the LD fraction after Iso treatment. The amount of TAG or ADRP was used to normalize sample loading. F, Fsp27 accumulation slightly increases in the ER fraction of 3T3-L1 adipocytes after Iso treatment. Calnexin (CNX) was used as a specific marker and loading control for the ER fraction. G, lipid droplet morphology and enhanced Fsp27 staining signal on the LDs of 3T3-L1 adipocytes in the presence of Iso (2 h). LDs were stained with BODIPY (green), and Fsp27 was stained with a rabbit anti-Fsp27 antibody (red). Scale bar, 5 μm.