Analysis of biogenesis of lipid droplets by examining Rab40c associating with lipid droplets

Abstract

The biogenesis of lipid droplets (LDs) is regulated by multiple proteins. Rab40c is a recently characterized small GTPase associating with LDs. Here we describe our approaches to analyze the involvement of Rab40c in the biogenesis of LDs. Co-localization of Rab40c and LDs was revealed through fluorescence confocal microscopy by expressing GFP-Rab40c and labeling LDs with dyes Oil Red O, Nile Red, or BODIPY 493/503. Association of Rab40c with LDs was further confirmed by fractionation with sucrose density centrifugation. The relationship of Rab40c with adipocyte differentiation was examined by detecting the protein level of Rab40c during adipocyte differentiation of 3T3-L1 cells. The roles of Rab40c in the biogenesis of LDs were analyzed by inducing LD formation in HepG2 cells.