Supplementary Material
Rational drug redesign to overcome drug resistance in cancer therapy:
imatinib moving target
Ariel Fernández1,2,3 *, Angela Sanguino3, Zhenghong Peng4,
Alejandro Crespo1, Eylem Ozturk3, Xi Zhang2,
Shimei Wang4, William Bornmann4 and Gabriel Lopez-Berestein3
(1) Rice University, Department of Bioengineering, Houston, TX 77005
(2) Division of Applied Physics, Rice University, Houston, TX77005
(3) Experimental Therapeutics, M. D. Anderson Cancer Center,
1515 Holcombe, Box 422, Houston, TX 77030
(4) Experimental Diagnostic Imaging, Chemistry Section, M. D. Anderson Cancer
Center, 1515 Holcombe, Box 603, Houston, TX 77030
Methods
Calculation of local dehydration propensities. The local mean residence time, <τ>i, of
hydrating molecules at residue i is defined with respect to a spherical domain D(i) of 6Åradius centered at the α-carbon of residue i. The actual computation of residence times is
given as:
<τ>i = ∫τfi(τ)dτ/(∫fi(τ)dτ), ∫0≤τ’≤τ fi(τ')dτ' = Pi(0)-Pi(τ),
Pi(τ) = Θ−1∫0≤t≤Θ[Σ
v(t)∈U(i,t) δ(v(t),
w(t+τ)∈U(i,t+τ)
w(t+τ))]dt ;
(1)
where fi(τ)dτ/∫fi(τ)dτ is the expected fraction of water molecules that remained in D(i)
for a period
τ and exit D(i) within a period of time in the range (τ, τ+dτ]; Pi(τ) is the
expected number of water molecules remaining in D(i) after a period τ has elapsed; v(t),
w(t+τ) denote indexes labeling water molecules contained in D(i) at times t and
1
t+τ, respectively; U(i,t), U(i,t+τ) denote collection of indexes of water molecules
contained in D(i) at times t and t+τ, respectively; δ is the Kronecker symbol (δ(v(t),
w(t+τ))=1 if v(t)=w(t+τ) and 0, otherwise); and the integration over variable t is carried
out over the interval of sampled times (t=0 to t=Θ=10ns) after 5ns of prior equilibration
(the sampling is considered exhaustive since <τ> << Θ for all residues).
Molecular dynamics/free-energy computations. Classical molecular dynamic (MD)
simulations were performed starting from the crystal structure of the C-KIT kinase
(PDB.1T46, 1.60 Å resolution) (1). Simulations were performed for the wild-type kinase
complexed with imatinib, as given by the crystal structure. We performed simulations of
the uncomplexed free form of the C-KIT kinase in order to correctly describe the induced
fit. To account for the effect of the drug resistance mutations, simulations were
performed for the in-silico generated C-KIT D816V mutant, in presence/absence of
imatinib and WBZ_7. Finally, similar simulations were performed for the isolated
inhibitors.
The initial structures were immersed in a pre-equilibrated truncated octahedral cell of
TIP3P explicit water molecules (2) and Cl– ions were added to neutralize the system
using the LEAP module of AMBER, version 9 (3-6). Protein atoms were described with
the parm99SB force field parameterization (7). Water molecules extended at least 8 Å
from the surface of the protein. The PMEMD module of AMBER was used to perform
the simulations in the NPT ensemble, employing periodic boundary conditions. Ewald
sums (8) and a 8 Å distance cutoff were used for treating long-range electrostatic
interactions. A SHAKE algorithm was employed to keep bonds involving hydrogen
atoms at their equilibrium length (9), which allowed us to employ a 2 fs time step for the
integration of Newton’s equations. Constant pressure of 1 atm and temperature of 300K
was maintained using the Berendsen coupling scheme (10). The optimized systems were
heated to 300 K and equilibrated for 200 ps. The resulting structures were the starting
point of the production 50 ns MD simulations.
2
For both inhibitor molecules, we used the variant structure reported by Kuriyan (11).
These structures differ from imatinib because they lack the piperazinyl group attached to
the phenyl-ring. The modification increases the solubility but does not alter their binding
significantly. The resulting inhibitors were parameterized using the ANTECHAMBER
module of AMBER with the GAFF force field parameterization (13). Charges of these
molecules were assigned conforming to the BCC charge model using AM1 optimized
geometries and potentials (13,14).
The free energies of binding for the different enzyme/inhibitor systems were calculated
using the MM-PB(GB)SA method (15-19), as implemented in the MM_PBSA module of
the AMBER package. In this method free energies are calculated for snapshot structures
taken from the MD trajectory of the corresponding enzyme/inhibitor complex. The
average binding free energy (∆Gbind) is calculated from the sum of the average molecular
mechanical gas-phase energies (EMM), the solvation free energies (∆Gsolv) and the entropy
contributions (–TS), as:
∆Gbind = ∆EMM +∆∆Gsolv -T∆Sbind
(2)
with ∆X = Xcomplex - Xfree – Xinhibitor. Complex, free and inhibitor correspond to the
complexed structure of the C-KIT kinase with the inhibitor, uncomplexed C-KIT kinase
and inhibitor alone, respectively. The snapshots were taken every 10 ps from the 50 ns of
the production MD, yielding a total of 5000 structures. The molecular mechanical (EMM)
energies, were evaluated in a single MD step in the SANDER module of the AMBER
package using an infinite cutoff for nonbonded interactions. The solvation free energies
(∆Gsolv) were estimated as the sum of an electrostatic solvation energy, calculated by the
generalized Born model (∆GGB) (20), plus a non-polar solvation energy (∆Gnp),
calculated from the solvent-accesible surface area (SASA):
∆Gsolv = ∆GGB + ∆Gnp
(3)
For the electrostatic solvation free energies we used a dielectric constant of 1 for the
solute and 80 for the aqueous solvent. We adopted the approximate pairwise method to
calculate the “effective” Born radius as described by Hawkins et al (21). The algorithm of
Sanner (22) was used to calculate the SASA for the estimation of the non-polar solvation
energy, ∆Gnp=γSASA, with a proportionality constant of 72 cal.mol-1.Å-2, as implemented
in the MSMS module of the AMBER package.
3
The entropic contributions to the binding free energy were estimated by calculating the
quasiharmonic entropy (23-26) from the MD trajectories with the PTRAJ module of the
AMBER package. The quasiharmonic entropy takes into the account both the
configurational and vibrational entropy contributions arising from structural changes and
creation of new modes upon complexation. The calculation of the quasiharmonic
entropies from MD trajectories were carried out using the approach developed by
Schlitter (27), which is based on the entropy of a quantum mechanical harmonic
oscillator. The entropy definition based on covariance matrix of atomic positional
fluctuations of the Cartesian coordinates of the particles in the system is given as:
⎡ k BTe 2
⎤
1
S = k B ln det ⎢1+ 2 Mσ ⎥
2
η
⎣
⎦
(4)
where kB is the Boltzmann constant, T is the temperature, e is the Euler value, ħ = h/2p; h
is Planck’s constant, and M is the mass diagonal matrix. The σ-value is the covariance
matrix of the atomic positional fluctuations with elements:
σ = <(xi - <xi>)(xj - <xj>)>
(5)
The approximation yields an upper limit of the classical entropy. Before calculating the
configurational entropy, the structures in the trajectory are spatially superposed to a
common reference structure to exclude all rotational and translational motions during the
simulation. The application of the covariance matrix calculation was performed selecting
the backbone atoms (N, Cα, C) of the protein structures and the heavy atoms (C, N, O)
for the inhibitors, as a reduced representation of the system, avoiding an estimate of the
entropy from the fluctuations of atoms within a residue. The 15 N- and C-terminal
residues were also excluded from the calculation due to their higher structural fluctuation,
which may yield in an overestimation of the entropy.
4
Total Synthesis of WBZ_7
The synthesis begins with treatment of 2-methyl-5-nitroaniline (1) with 65% nitric acid
in ethanol followed by the addition of cyanoamide to give the corresponding 2-methyl-5nitroaniline-guanidine nitrate (2). One completed, 3-Acetyl-pyridine (3) was treated with
N,N-dimethyl acetamide dimethyl acetal to give 3-dimethylamino-2-methyl-1-(3pyridyl)-2-propene-1-one (4). The nitrate salt (2) is treated with (4) and sodium
hydroxide in refluxing n-butanol to give N-(2-Methyl-5-nitrophenyl)-5-methyl-4-(3pyridyl)-2-pyrimidine-amine (5) which is subsequently reduced with stannous chloride to
give N-(2-Methyl-5-aminophenyl)-5-methyl-4-(3-pyridyl)-2-pyrimidine-amine (6). The
WBZ7 synthesis will consist of the reaction of α-chloro-p-toluylic acid (7) with 4methyl-piperazine in ethanol followed by treatment with con. HCl to give the
corresponding dihydrochloride 4-(4-methyl-piperazin-1-ylmethyl)-benzoic acid (8) which
is subsequently treated with thionyl chloride to give the corresponding acid chloride
dihydrochloride (9). Subsequent condensation with N-(2-Methyl-5-aminophenyl)-4-(3(6-methyl)-pyridyl)-2-pyrimidine-amine (6) in pyridine affords the imatinib analog
WBZ_7 (28).
Material and Methods: All chemicals and solvents were obtained from Sigma-Aldrich
(Milwaukee, WI) of Fisher Scientific (Pittsburg, PA) and used without further
purification. Analytical HPLC was performed on a Varian Prostar system, with a Varian
Microsorb-MW C18 column (250 X 4.6 mm; 5µ) using the following solvent system A=
H2O /0.1% TFA and B=acetonitrile/0.1% TFA. Varian Prepstar preparative system
5
equipped with a Prep Microsorb–MWC18 column (250 X 41.4 mm; 6µ; 60 Å) was used
for preparative HPLC with the same solvent systems. Mass spectra (ionspray, a variation
of electrospray) were acquired on an Applied Biosystems Q-trap 2000 LC-MS-MS. UV
was measured on Perkin Elmer Lambda 25 UV/Vis spectrometer. IR was measured on
Perkin Elmer Spectra One FT-IR spectrometer. 1H-NMR and
13
C-NMR spectra were
recorded on a Brucker Biospin spectrometer with a B-ACS 60 autosampler. (600.13 MHz
for 1H-NMR and 150.92 MHz for 13C-NMR), Chemical shifts (δ) are determined relative
to d4-methanol (referenced to 3.34 ppm (δ) for 1H-NMR and 49.86 ppm for 13C-NMR).
Proton-proton coupling constants (J) are given in Hertz and spectral splitting patterns are
designated as singlet (s), doublet (d), triplet (t), quadruplet (q), multiplet or overlapped
(m), and broad (br). Flash chromatography was performed using Merk silica gel 60 (mesh
size 230-400 ASTM) or using an Isco (Lincon, NE) combiFlash Companion or SQ16x
flash chromatography system with RediSep columns (normal phase silica gel (mesh size
230-400ASTM) and Fisher Optima TM grade solvents. Thin-layer chromatography
(TLC) was performed on E.Merk (Darmstadt, Germany) silica gel F-254 aluminumbacked plates with visualization under UV (254nm) and by staining with potassium
permanganate or ceric ammonium molybdate.
2-methyl-5-nitrophenyl-guanidine nitrate(2) (29). 2-Methyl-5-nitroaniline (100 g,
0.657 mol) was dissolved in ethanol (250 ml), and 65% aqueous nitric acid solution (48
ml, 0.65 mol) was added thereto. When the exothermic reaction was stopped, cyanamide
(41.4 g) dissolved in water (41.4 g) was added thereto. The brown mixture was reacted
under reflux for 24 hours. The reaction mixture was cooled to 0° C., filtered, and washed
with ethanol:diethyl ether (1:1, v/v) to give 2-methyl-5-nitrophenyl-guanidine nitrate (98
g). Rf=0.1 (Methylene chloride:Methanol:25% Aqueous ammonia=150:10:1). MS: 195.2
(M+H); 1H-NMR(DMSO-d6)=1.43(s, 3H), 6.59(s, 3H), 6.72-6.76(d, 1H), 7.21-7.27(m,
1H), 8.63-8.64(br, 1H).
3-dimethylamino-2-methyl-1-(3-pyridyl)-2-propen-1-one(4). 3-Acetyl-pyridine (9g, 75
mmol) was added to N,N-dimethyl acetamide dimethyl acetal (11 ml, 75 mmol), and the
mixture was reacted under reflux for 18 hours. After the reaction mixture was cooled to
0° C., The solution was evaporated to dryness and a mixture of diethyl ether and hexane
(3:2, v/v) (10 ml) was added and the whole mixture was stirred for 4 hours. The resulting
6
solid was filtered and washed with a mixture of diethyl ether and hexane(10 ml, 3/2, v/v)
to give 3-dimethylamino-2-methyl-1-(3-pyridyl)-2-propen-1-one (10 g, 50 mmol, 70%).
Rf=0.46 (Methylene chloride:Methanol=9:1). MS: 191.1 (M+H);
9.00 (s, 1H), 8.61 (d, J= 4.2H, 1H), 8.15(d, J= 7.8 Hz, 1H),
1
H NMR(DMSO) δ
7.43 (dd, J= 7.8, 4.8 Hz,
1H), 5.64 (s, 1H), 3.08 (s, 6H), 2.60 (s, 3H); 13C NMR δ 183.40, 164.43, 150.64, 148.20,
137.65, 134.39, 123.26, 90.79, 16.00.
N-(2-Methyl-5-nitrophenyl)-5-methyl-4-(3-pyridyl)-2-pyrimidine-amine
(5).
3-
dimethylamino-2-methyl-1-(3-pyridyl)-2-propen-1-one (4) (3.9g, 21 mmol), 2-methyl-5nitrophenyl-guanidine nitrate (2) (5.3 g, 20 mol), and sodium hydroxide (900 mg, 23
mmol) were dissolved in n-butanol 100 ml and reacted under reflux for 18 hours. The
reaction solution was cooled to 0° C., filtered, washed with isopropanol and methanol,
and dried to give N-(2-methyl-5-nitrophenyl)-5-methyl-4-(3-pyridyl)-2-pyrimidineamine. 5g 76%. MS 322.5 (M+H). 1H NMR(DMSO) δ 9.31 (1H), 9.21 (1H), 8.90 (1H),
8.71(1H), 8.47(1H), 7.88(1H), 7.52 (3H), 2.46 (s, 3H), 2.43 (s, 3H).
N-(2-Methyl-5-aminophenyl)-5-methyl-4-(3-pyridyl)-2-pyrimidine-amine (6). The
above N-(2-methyl-5-nitrophenyl)-5-methyl-4-(3-pyridyl)-2-pyrimidine-amine (2.2 g, 6.8
mmol) was suspended in water 25 ml and 25 ml of concentrated hydrochloric acid. To
this stannous chloride 7.5 g was added ant heated at 50oC for 2 hours. The reaction was
cooled down to r.t. and neutralized with sodium hydroxide. The solid was filtered and the
solid was extracted with ethyl acetate. The organic layer was concentrated to give the title
compound. MS: 292.2 (M+H); 1H NMR(DMSO) δ 9.22 (d, 1H), 8.68 (d, J= 4.8 Hz, 1H),
8.55 (s, 1H), 8.38 (d, , J= 8.1 Hz, 1H), 7.53 (dd, J= 8.0, 5.4 Hz, 1H), 7.29 (s, 1H),
6.87(d, J= 8.1 Hz, 1H), 6.84(s, 1H), 6.33 (dd, J= 8.0, 5.4 Hz, 1H), 4.84 (s, 2H), ), 2.38
(s, 3H), 2.07 (s, 3H); 13C NMR δ 168.78, 161.15, 151.08, 148.05, 146.67, 138.10,134.18,
132.48, 130.05, 123.74, 119.09, 110.85, 110.65, 106.52, 23.86, 17.26.
4-(4-methylpiperazinomethyl)benzoic acid dihydrochloride (8) (30). To a well-stirred
suspension consisting of 17.1 g. (0.10 mole) of α-chloro-p-toluylic acid in 150 ml. of
absolute ethanol under a nitrogen atmosphere at room temperature (~20° C.), a solution
consisting of 44.1 g. (0.44 mole) of N-methylpiperazine dissolved in 50 ml. of ethanol
was added dropwise. The resulting reaction mixture was refluxed for a period of 16 hours
and then cooled to room temperature. The cooled reaction mixture was concentrated in
7
vacuo and the thus obtained residue partitioned between 100 ml. of diethyl ether and 100
ml. of 3N aqueous sodium hydroxide. The separated aqueous layer was then washed
three times with 100 ml. of diethyl ether, cooled in an ice-water bath and subsequently
acidified with concentrated hydrochloric acid. The resulting solids were filtered and airdried, followed by trituration with 150 ml. of boiling isopropyl alcohol and stirring for a
period of two minutes. After filtering while hot and drying the product there were
obtained 9.4 g. (35%) of pure 4-(6-methylpiperazinomethyl)benzoic acid dihydrochloride
as the hemihydrate, m.p. 310°-312° C. MS: 235.1 (M+H); 1H NMR(D2O) δ 8.04 (d, J=
8.21 Hz, 2H), 7.59 (d, J= 8.21 Hz, 2H), 3.50 (s, 2H), 3.63 (br, 8H), 2.97 (s,3H); 13C NMR
δ 170.18, 133.13, 131.91, 130.90, 60.22, 50.61, 48.77, 43.25.
4-(4-methylpiperazinomethyl)benzoyl chloride dihydrochloride (9) (30). To 20 g.
(0.065 mole) of 4-(4-methylpiperazinomethyl)benzoic acid dihydrochloride under a
nitrogen atmosphere, there were added 119 ml. of thionyl chloride (194 g., 1.625 mole) to
form a beige-white suspension. The reaction mixture was refluxed for 24 hours and then
cooled to room temperature (~20° C.). The resulting suspension was filtered, and the
recovered solids were washed with diethyl ether and dried to ultimately afford 17.0 g.
(81%) of pure 4-(4-methylpiperazinomethyl)benzoyl chloride dihydrochloride.
N-{5-[4-(4-methyl piperazine methyl)-benzoylamido]-2-methylphenyl}-5-methyl-4[3-pyridyl]-2-pyrimidine amine (free base) WBZ_7. A mixture of N-(2-Methyl-5aminophenyl)-5-methyl-4-(3-pyridyl)-2-pyrimidine-amine (6) 250 mg (0.85 mmol) and
4-(4-methylpiperazinomethyl)benzoyl chloride dihydrochloride (9) 325 mg (1 mmol)
were stirred in 20 ml anhydrous pyridine at 20 oC for 18 hours. The reaction mixture was
concentrated in vacuum. The residue was subjected to silica gel chromatography using
5% Methanol (7M NH3 ) in DCM. MS: 508.5 (M+H); 1H NMR(CDCL3) δ 9.21 (s,1H),
8.74 (S,1H), 8.65 (d, J= 4.2 Hz, 1H), 8.5. (d, J= 7.8 Hz, 1H), 7.95 (s, 1H), 7.83(d, J= 8.4
Hz, 2H), 7.43 (d, J= 8.4 Hz, 2H), 7.38 (dd, J= 7.8, 4.8 Hz, 1H), 7.23 (d, J= 7.8 Hz, 1H),
7.17 (d, 7.8 Hz,1H), 7.05 (s,1H), 6.99 (s,1H), 3.56 (s, 2H), 2.50 (s, 3H), 2.50 (br, 8H),
2.34 (s,3H), 2.29 (s,3H); 13C NMR δ 169.20, 165.39, 162.34, 160.39, 151.21, 148.47,
142.54, 138.16, 136.62, 125.10, 134.03, 132.99, 130.64, 129.31, 127.01, 123.70, 123.60,
114.78, 112.75, 108.03,62.53,55.13, 53.15,46.03,24.41, 17.71.
8
H
N
N
N
H
N
N
CH3
O
N
WBZ7
N
Alternative substitutions at the indicated position with ethyl, propyl and isopropyl
groups were attempted but they proved to be ineffective to yield any detectable inhibition
of the D816V mutant or even the wild-type kinase. The likely cause of such lack of
activity is the steric clash between the ligand substituting group and the side chain of
F811 (Fig. 1b).
Spectrophotometric kinetic assays. Reactions were carried out at 35ºC in 500µl of
buffer (100mM Tris-HCl, 10mM MgCl2, 0.8mM ATP, 1mM phosphoenol pyruvate,
0.33mM NADH, 95 units/ml pyruvate kinase, pH7.5). Maximum rates of downstream
phosphorylation were measured at initially saturating ATP concentration. The kinetics
assays for each kinase/ligand pair were repeated five times, at 100nM inhibitorconcentration
increases.
The
substrate
adopted
(Invitrogen/Biaffin)
is
KVVEEINGNNYVYIDPTQLPY (31) and is assayed at 2mM, a saturating concentration
for the 10nM initial kinase concentration. Hence, within relevant timescales, fluctuations
in the concentration of the substrate-kinase complex do not interfere with the
phosphorylation kinetics subject to ATP-competitive inhibition. These conditions enable
us to describe the kinetics with two effective variables, [ATP] and [I]=inhibitor
concentration. The kinetics of downstream phosphorylation were fitted following a
Michaelis-Menten scheme with three components: a) initial saturating ATP-concentration
yielding a maximum initial phosphorylation velocity, Vo=Vmax; b) ATP-competitive
inhibitor that binds reversibly; and c) ATP-binding equilibrium constants Km≈18±2µM
for the wild-type C-Kit kinase, Km≈31±2µM for the D816V mutant, obtained in the
absence of inhibitor. These Michaelis-Menten constants were obtained from the
respective ATP-concentrations that yielded Vo=Vmax/2. The apparent Michaelis-Menten
9
constants, Km(app)s, are given by Km(app)=Km(1+[I]/Kd), with Kd=inhibitor-kinase
dissociation constant. Phosphorylation rate curves were fitted to the equation
Vo=Vmax[ATP]/{Km(app)+[ATP]}, and thus enable direct inference of Kd.
Cell proliferation assay. GIST cancer cells (line ST882) expressing C-Kit kinase (32)
were seeded in 96 well plates (8x103 cells per well) in 100 µl of RPMI-1640 medium and
cultured for 24h. Four groups of batteries of 24 cell assays were conducted and each
group of assays entailed treatment with a different in-bulk concentration (10, 100, 1000
and 104 nM) of imatinib. An identical battery of 96 cell assays divided into 4 groups were
conducted for WBZ_7. Cell proliferation was determined by Alamar Blue assay (Bio
Source International, Camarillo, CA). Following 48h of exposure, 50 µl of medium was
removed from each well and place into a new 96-well plate. To reach a final volume of
100 µl per well, 40 µl of fresh media and 10 µl of Alamar Blue probe were added. Plates
were read at dual wavelength (570-595 nm) in an Elisa plate reader (Molecular Devices,
Sunnyvale). The battery of 24 assays for each inhibitor concentration generated a mean
value and dispersion in the percentage of cell proliferation with respect to a normalized
control exposed to zero inhibitor concentration.
Proliferation of treated murine pro-B cells Ba/F3 (ATCC, Manassas, VA)
expressing C-Kit D816V (33) was investigated using a tetrazolium-based assay (34)
adopting the same generic set-up described above. Cell proliferation was determined
spectrophotometrically by measuring the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium (MTT) activity following a 48h treatment with imatinib and WBZ_7.
Human HMC-1 mast cells (35) (gift from J.H. Butterfield, Mayo Clinic),
expressing the C-Kit D816V-mutant were cultured in Iscove's modified Dulbecco's
medium (Mediatech Inc., Herndon, VA) supplemented with 10% (v/v) iron-supplemented
fetal calf serum (FCS) (Gibco BRL) and 1.2 mM α-thioglycerol (Sigma, St Louis, MO,
1
USA) at 37°C under 5% CO2 in a humidified incubator. HMC-1 cells were seeded in 96
wells plates at a density of 3 x104 cells per well in 50 µl of medium. Two hours later, 50
µl medium containing different concentrations of imatinib and WBZ_7 were added to the
wells to reach a final volume of 100 µl per well. Following 48h of exposure, an MTS
assay
[3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-
tetrazolium, inner salt] was performed. We mixed 100 µl of phenazine methosulfate
(PMS) (0.92 mg/ml) (Sigma, St Louis, MO) for every 2 ml of MTS (2 mg/ml) (Promega,
Madison, WI). We added 20 µl of the MTS/ PMS mixture to each well and incubate at 37
C for 30 minutes. Plates were read at 490 nm.
Western blots. After incubation with inhibitors, cell pellets were lysed using a 1%
Triton-X buffer (1% Triton-X, 150 mM NaCl, 25 mM Tris, pH 7.6) to which 4mM
sodium fluoride (NaF) and 4 mM sodium orthovanadate were added. Total protein
concentration was determined using a DC protein assay kit (Bio-Rad, Hercules, CA).
Aliquots containing 50 µg of total protein from each sample were subjected to sodium
dodecyl sulfate-polyacrilamide gel electrophoresis (SDS-PAGE). The membranes were
blocked with 5% dry milk in TBST (100 mM Tris-HCL [pH 8.0], 150 mM NaCl, and
0.05% Tween-20), probed with primary antibodies diluted in TBST containing 5% dry
milk, and incubated at 4°C overnight. We used primary antibodies against phospho-c-kit
(Tyr703) (Zymed Laboratories Inc., San Francisco, CA), anti-c-kit (E-1), (Santa Cruz
Biotechnology Inc, CA) and anti-β-actin (Sigma, St. Louis, MO). Rabbit and anti-mouse
secondary antibodies were purchased from Cell Signalling, Danvers, MA. Protein bands
were visualized by enhanced chemiluminescence reagent plus (PerkinElmer, Wellesley,
MA). Images were analyzed densitometrically using the Alpha Imager application
program (Alpha Innotech, San Leandro, CA).
1
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