Biomedical Engineering Reference
In-Depth Information
of this organelle but also signifi cant for designing drugs to treat
LDs-related diseases. Although LDs are most likely derived from
endoplasmic reticulum (ER), there are specialized proteins associ-
ated with LDs. Since these are lipid storage organelles, there are
enzymes for lipid metabolism associating with LDs. In particular,
there are special proteins associated with the membrane of LDs to
regulate the biogenesis of LDs. The PAT family proteins [perilipin,
ADRP (adipocyte differentiation related protein or adipophilin) and
TIP47 (tail-interacting protein 47 kDa)] are the best characterized
proteins crucial for the biogenesis of LDs [
4
-
9
]. Proteomic investi-
gations have also discovered that the protein components of LDs
vary depending on cell types or physiological conditions [
10
-
13
].
SNARE and Rab proteins are key regulator for membrane traffi ck-
ing in eukaryotes. Some Rab proteins have been reported to be
involved in the biogenesis of LDs [
14
,
15
]. Although specifi c
SNARE complex mediating LDs traffi cking have not yet been iden-
tifi ed, SNARE proteins may be involved in regulating LDs mem-
brane fusion [
16
].
The roles of some LD-associated proteins in regulating the
biogenesis of LDs have been examined by immuno-electron
microscopy, immunofl uorescence microscopy, biochemical meth-
ods, as well as in animal models [
17
,
18
]. Our previous work has
demonstrated that Rab40c is a novel LDs associated Rab protein
[
19
]. Here we describe the approaches applied to identify the LD
association of Rab40c, including fl uorescence confocal micros-
copy, LD fractionation, stimulation of adipocyte differentiation
with 3T3-L1 cells, and induction of biogenesis of LD in HepG2
cells with oleic acid. The methods described here would be useful
in general for the characterization of proteins associating with LDs,
particularly other Rab proteins.
2
Materials
2.1 Cell Culture
and Transfection
1. NRK (Normal rat kidney), 3T3-L1 and HepG2 cell lines
were from the American Type of Culture Collection (ATCC)
(
see
Note 1
).
2. DMEM medium (4,500 mg/L glucose, Gibco) containing
10 % (v/v) fetal bovine serum (FBS, Hyclone) or 10 % fetal calf
serum (FCS, Hyclone).
3. Transfection reagent: Lipofectamine 2000 (Invitrogen).
4. Trypsin solution: 0.25 % trypsin in PBS containing 1 mM
EDTA.
GFP-Rab40c was constructed by inserting the coding sequence of
human Rab40c cDNA into the EcoRI/SalI sites of pEGFP-C1
vector (BD Clontech) [
19
].
2.2 Expression
Plasmids
