and cells were passaged every 10
-
14 days. The neurospheres were cultured for 6
-
10 weeks in 5% CO
2
at 37
°
C before use.For inhibition of GSK3
β
, cell cultures were incubated for 3days in the presence of 10
µ
M (final concentration) 3-(2,4-dichlorphenyl)-4-(1-methyl-1
H
-indol-3-yl)-1
H
-pyrrole-2,5-di-one (SB216763; Tocris, Ellisville, MO), a specific low-molecular weight inhibitor of GSK3.
13
-
15
Five cell cultures were used ineach group for comparative replicates, run in individual gels.
Two-Dimensional Gel Electrophoresis (2-DE)
.
2-DE wasperformed using standard protocols as previously described.
11,12,16
Cells were harvested, and protein extraction for 2-DE wasperformed for 60 min at room temperature in a lysis buffercontaining 7 M urea, 2 M thiourea, 4% (w/v) CHAPS, 0.5% (v/v) Triton X-100, 100 mM DTT, 0.05% IPG buffer, pH 3
-
10(Amersham Biosciences, Uppsala, Sweden), and 0.156% (w/v)Complete protease inhibitor tablets (Roche, Mannheim, Ger-many). Sample protein amounts were determined by themodified Bradford method.
17,18
A total of 250
µ
g (5
-
10
µ
L) of the protein solution was suspended in rehydration solutionconsisting of 6 M urea, 2 M thiourea, 2% (w/v) CHAPS, 0.5%(v/v) IPG buffer, pH 3
-
10, and a few grains of bromophenolblue to give a final volume of 350
µ
L. The samples were appliedto pH 3
-
10 nonlinear gradient IEF gel strips for isoelectricfocusing in the IPGphor apparatus (Amersham Biosciences,Uppsala, Sweden). The IEF gel strips reswelled for 12 h at 30 V to remove high salt concentrations and to improve proteinentry into the gel. Then 200, 500, and 1000 V were applied for1 h each. Voltage was increased to 8000 V in 30 min and keptconstant at 8000 V for 12 h, resulting in a total of 100 300 Vh.Gel strips were equilibrated for 20 min each in an SDSequilibration buffer consisting of 50 mM Tris-HCl, pH 8.8, 6 Murea, 30% (v/v) glycerol, 2% (w/v) SDS, a few grains of bromophenol blue, and 1% (w/v) dithiothreitol or 2.5% (w/v)iodoacetamide, respectively. The second-dimension separation was performed using 12.5% polyacrylamide gels in the presenceof 0.1% (w/v) sodium dodecylsulfate. The gels were run at 30mA for 30 min and 100 mA for about 4 h in a 20 cm
×
20 cm water-cooled vertical electrophoresis apparatus (OWL, Woburn,MA). For image analysis, gels were stained by the “Blue silver”method, a sensitive Coomassie-based quantitative stain.
19
Briefly, gels were soaked overnight in 0.12% Coomassie BlueG-250, 10% phosphoric acid, 10% ammonium sulfate, and 20%methanol and destained the next day for 5
-
6 h.
Gel Image Analysis and Mass Spectrometry
.
Gels werescanned, and images were analyzed using the Phoretix 2DExpression software (Nonlinear Dynamics, Newcastle-upon-Tyne, U.K.). Image analysis was performed as described.
20
Normalized spot volumes defined as integral of spot areamultiplied by optical densities were compared to normalizedmeans
(
standard deviations from 3 gels of each group by Student’s
t
test for unpaired data.
21
Spots of interest wereexcised and digested by trypsin for mass spectrometry (Centrefor Molecular Medicine, ZMMK, University of Cologne, Ger-many), and mass spectra were identified by searching the NCBInonredundant protein database with Mascot
22
(http:// www.matrixscience.com) as described elsewhere in detail.
23
Briefly, trypsinized protein samples were loaded onto prespot-ted AnchorChip targets which are stainless steel supportscoated with hydrophobic material equipped with an array of 384 circular interruptions (anchors) of 600 mm diameter(Bruker-Daltonics, Bremen, Germany). They were preparedusing
R
-cyano-4-hydroxycinnamic acid (HCCA) as matrix, whereby 0.3 mL of analyte solution and 1.2 mL of matrix solution (0.3 g/L HCCA in ethanol/acetone
)
2:1) were appliedonto the anchors using an Investigator ProMS MALDI Spotting Robot (Genomic Solutions, Ann Arbor, MI). Samples wereallowed to air-dry at room temperature. Peptide mass spectra were obtained using an Ultraflex TOF/TOF (Bruker-Daltonics)in the fully automated reflectron TOF mode operated by theflexControl software. The mass spectrometer was equipped witha SCOUT-MALDI source for multisample handling, a pulsedUV laser, a two-stage gridless reflector, a 2 GHz digitizer, a LIFT-TOF/TOF unit to analyze fragment ions of selected peptide ions(see below), and multichannel-plate detectors for linear andreflector mode measurements. All measurements were carriedout in positive ionization mode using a reflector voltage of 20kV. The external instrument calibration was achieved using signals from [M 1 H]1 ions of the following reference standards(
m
/
z
): Bradykinin clip (1
-
7) mono 757.39916; Angiotensin IImono 1046.5418; Angiotensin I mono 1296.6848; Substance Pmono 1347.7354; Bombesin mono 1619.8223; Renin substratemono 1758.93261; adrenocorticotropic hormone (ACTH) clip(1
-
17) mono 2093.0862; ACTH clip (18
-
39) mono 2465.1983;and Somatostatin clip (28) mono 3147.4710. Fragment
m
/
z
spectra were obtained by integration over up to 2000 successivelaser pulses (
f
)
50 Hz). The spectra were calibrated using autodigestion peptide signals for trypsin (
m
/
z
842.5094,1045.5637, 2211.1040, and 2283.1802) as reference values and were the basis of mining the NCBI nonredundant database forprotein identification via Mascot query (Matrix Science, Lon-don, U.K.) with the following parameters: enzyme, trypsin;missed cleavages, 1; allowed modifications, carbamidomethyl(fixed) and methionine oxidation (variable); tolerance, 75 ppm,that is, mass measurement accuracies were typically
(
75 ppm.The Mascot-delivered probability based score was regarded asa quality parameter for the correct identification.
22
Protein spotsthat could not successfully be identified with the previousmethod were additionally analyzed by peptide sequencing withthe same device, Ultraflex MALDI LIFT-TOF/TOF. Therefore,a high-resolution timed ion selector to separate selectedpeptide ions, a LIFT device for raising the potential energy of fragment ions, a velocity focusing stage with subsequentpostacceleration, and a postLIFT metastable suppressor device were used. For the analysis, up to five precursor ions wereselected. The ions were subjected initially to acceleration with8 kV in mass spectrometer step one, then selected using a timedion-gate, and finally energy-lifted to a voltage of 19 kV.Fragmentized ion species were then accelerated in the secondion source and analyzed in mass spectrometer step two,running in reflector mode. Mascot analysis of the obtainedspectra was performed with 0.8 Da tolerance, one missedcleavage, and carbamidomethyl and methionine oxidation asallowed modifications.
Reverse Transcription-Polymerase Chain Reaction (RT-PCR).
Neurospheres were harvested for total RNA extractionusing the RNeasy kit (Qiagen, Hilden, Germany). RNA wasreversely transcribed using the Omniscript RT kit (Qiagen,Hilden, Germany). Specific cDNA target sequences for GlycogenSynthase Kinase 3
β
(GSK3
β
; NM_032080), forward, 5
′
-GGATCTGCCATCGAGACATT-3
′
; reverse, 5
′
-CCAACTGATCCA-CACCACTG-3
′
; Wnt5a (NM_022631), forward, 5
′
-TGGAGTGG-TAAATGCCATGA-3
′
; reverse, 5
′
-ATACTGTCCTGCGACCTGCT-3
′
; Wnt7a (XM_342723), forward, 5
′
-CCCGAACCCTCATGAACTTA-3
′
; reverse, 5
′
-TAGCCTGAGGGGCTGTCTTA-3
′
; and Bone Mor-phogenic Protein 4 (BMP4; NM_012827), forward, 5
′
-CC-TGGTAACCGAATGCTGAT-3
′
; reverse, 5
′
-TCCTCACAGTGTTG-
GSK3
β
Regulates Neural Stem Cell Differentiation
research articles
Journal of Proteome Research
•
Vol. 6, No. 3, 2007
1199
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