Biology Reference
In-Depth Information
4
C with continuous gentle inversion. After the agarose beads are washed with lysis
buffer three times, the 50
m
l of SDS-PAGE sample buffer is added to the agarose
beads. Fifteen microliters of supernatant is applied onto 5-12% gradient SDS-PAGE,
and the resolved proteins are analyzed on a Western blot using the antibody against
a
-tubulin. The membrane is stripped and reblotted with antibody against G
a
s to show
sample loading (
Fig. 12.6
A).
12.1.4.1.2
Immunocytochemistry
To reveals colocalization between activated Gs
a
and tubulin. PC12 cells are grown in
Dulbecco's-modified Eagle's medium (Invitrogen) supplemented with 10% fetal bo-
vine serum, 5% horse serum, and 1% antibiotic (penicillin and streptomycin). Cells are
maintained in a 5%CO
2
incubator at 37
C. Medium is changed every 3 days, and cells
are passaged once per week. PC12 cells in 12-well culture plates are transfected with
G
a
s-GFP using GenePORTERTM transfection reagent (Gene Therapy Systems, Inc.,
San Diego, CA, USA). The cells are treated with cholera toxin or vehicle control,
washed twice with PBS, and fixed with cold 100%methanol for 4 min after extraction
with 0.2% (w/v) saponin in microtubule-stabilizing buffer. The coverslips are then in-
cubated with PBSS buffer (PBS plus 0.01% saponin) containing 10% bovine serum
albumin for 20 min and then incubated in a 1:1000 dilution of anti-
a
-tubulin in PBSS
buffer for 3 h. Subsequently, the coverslips are washed with PBSS four times and in-
cubated with a 1:180 dilution of secondary antibodies labeled with TRITC in PBSS
buffer for 40 min. These coverslips are washed with PBSS buffer four times and
mounted on the slide with mounting medium. The slides are air-dried and examined
by deconvolution microscopy. Images are captured with the Applied Precision, Inc.
(Seattle, WA, USA) DeltaVision system built on an Olympus IX-70 base. Z-stacks
are then deconvolved using the Softworx software. To quantify the colocalization be-
tween G
a
s and microtubules, images are imported to Volocity (Improvision Inc., Wal-
tham, MA, USA) for colocalization analysis. The extent of overlap between G
a
sand
microtubules is defined with Pearson's correlation.
12.1.4.2
Microtubule dynamics assay in cells
By using detergent-extracted cytoskeletons under microtubule-stabilizing condi-
tions, cellular microtubule mass can be measured in the absence of unassembled tu-
bulin. Activated Gs
a
decreases the stable pool of polymerized microtubules in intact
cells. In addition, using antibodies specific to different type of tubulin posttransla-
tional modification demonstrated that activated Gs
a
also reduces the level of
dynamic microtubules (acetylated MT) in the cells.
12.1.4.2.1
Measurement of detergent-insoluble microtubules
To measure microtubule mass, detergent-extracted cytoskeletons, free of unassembled
tubulin, are prepared under microtubule-stabilizing conditions (
Drubrin, Feinstein,
Shooter, & Kirschner, 1985; Solomon, Magendantz, & Salzman, 1979
). The tubulin
content of the cytoskeletons is measured with immunoblotting. In brief, cells in a
25-ml flask are washed once with 37
C PBS and once with extraction buffer. Cells
