Biomedical Engineering Reference
In-Depth Information
Fig. 3
Analysis of adipocyte differentiation. (
a
) 3T3-L1 cells were induced to differentiate into adipocytes for
the indicated times, and processed for confocal microscopy. Lipid droplets were revealed by the dye BODIPY
493/503. (
b
) 3T3-L1 cells were induced to differentiate into adipocytes. The cells were lysed and subjected for
western blot to detect Rab40c and the markers Perilipin, ADRP, or TIP47 for lipid droplets.
-tubulin serves as
loading control. The results indicated that the level of Rab40c increased following with the adipocyte differen-
tiation. Bar = 20
ʲ
ʼ
m. Figure (
b
) is reproduced from the open access journal PLoS One [
19
]
4
Notes
1. NRK cells give a better vesicular structure of Rab40c in our
experiments. 3T3-L1, the pre-adipose cell line, was usually
used to induce adipocyte differentiation [
12
], and HepG2 cells
were used to mimic LD formation in adipocytes by stimulation
with oleic acids [
20
].
2. PolyAb against Rab40c has been described previously [
19
].
PolyAb against TIP47 (AnaSpec), Rabbit mAb against
Perilipin (Cell Signaling Technology), and polyAb against
ADRP (Santa Cruz) serve as markers for lipid droplets. mAb
against
ʲ
-tubulin (Sigma) serves as loading control. mAb
against KDEL receptor (BD Biosciences) serves as marker for
endoplasmic reticulum.
3. Oleic acid must be pre-complexed with fat free BSA to prevent
detergent effects of the unbound fatty acids as described [
21
].
When pipetting oleic acid, the tips of pipette should be cut off
