Supplementary two hours prior to transfection. MSCV-IRES-GFP EV,

Supplementary Methods
Reagents
RPMI with (#31870017) and without – (#89984) lysine and arginine, fetal bovine serum (FBS), Lglutamine,
and antibiotics were purchased from Invitrogen (Carlsbad, CA). SILAC amino acids, 13C6-Lysine
and 13C6-Arginine, were obtained from Cambridge Isotope Laboratories (Andover, MA). TPCK-treated
trypsin was from Worthington Biochemical Corp. (Lakewood, NJ). For Immunoaffinity purification of
phospho-tyrosine peptides, anti-phosphotyrosine rabbit monoclonal antibody (P-Tyr-1000) beads were
obtained from Cell Signaling Technology (Danvers, MA). Ibrutinib (PCI 32765) (# S2680) was purchased
from SellekChem (Houston, TX). All other reagents used in this study were from Fisher Scientific
(Pittsburgh, PA).
Cell culture
BaF3 cells were maintained in RPMI supplemented with 10% FBS and 100 IU/ml penicillin, 100
?g/ml streptomycin and 2mM L-glutamine. BaF3 cells were grown in RPMI with 10% WEHI conditioned
media as a source of IL3, given that these cells are IL3 dependent for their growth. BaF3 cells were
passaged to 0.3 million cells/mL every 2 days.32D cells were cultured in IMDM medium supplemented
with 10% FBS and 100 IU/ml penicillin, 100 ?g/ml streptomycin, 2mM L-glutamine and recombinant
murine IL3 (Miltenyi Biotechnology). 32D cells were passaged to 0.3 million cells/mL every 2 days.
CSF3R Retroviral Vectors
Retroviral MSCV-IRES-GFP (MIG) constructs encoding the WT and mutant GCFR were kindly
provided by Dr. Mohammad Azam at Cincinnati Children’s Hospital Medical Center. The experimental
details and validation of the cloning and mutagenesis were previously reported (1).
Retrovirus Production, transduction, and generation of G-CSFR expressing stable cell lines
Lenti-X 293T cells (Clontech) were seeded at 4×106
cells/10cm dish in DMEM (ThermoFisher
Scientific) supplemented with 10% heat-inactivated FBS (Atlanta Biologics), Penicillin-Streptomycin
(ThermoFisher Scientific), and L-Glutamine (ThermoFisher Scientific). The following day, cells were
replenished with fresh Opti-MEM GlutaMAX media (ThermoFisher Scientific) supplemented with 10%
FBS (Atlanta Biologics) at least two hours prior to transfection. MSCV-IRES-GFP EV, CSF3R, CSF3R-T618I,
and CSF3R-Q741X retroviruses were each individually produced by co-transfecting 9 ?g retroviral
vectors with 9 ?g retroviral packaging and envelope plasmid, pCL-Eco, into the 293T cells using TransitLT1
(Mirus) according to manufacturer instructions. Growth medium was changed the following day.
Virus-containing supernatant was collected 48 and 72 hours post-transfection, and either used
immediately or aliquoted and frozen at -80°C. Transduction of BaF3 or 32D cells was performed by
incubating cells in viral supernatant overnight at 37°C, 5% CO2. The transduction protocol was repeated
again the next day. Cells were sorted for GFP+
expression at 48 hours post-transduction.
Flow cytometry and cell sorting
All flow cytometric staining, sorting, and analysis was performed in 1X FACS Buffer (1% FBS,
0.01% NaN3 in DPBS). Cell sorting was performed on a MoFloXDP (Beckman Coulter, Brea, CA) or BD
FACSAria II with a 100 µm nozzle. Sorted populations of interest were collected in DPBS buffer
containing 50% FBS (Atlanta Biologicals). Flow cytometric analyses were performed on a FACS LSRII or
LSRFortessa (Becton, Dickinson and Company, Franklin Lakes, NJ). Data were analyzed with FlowJo
Software (TreeStar, Ashland, OR).
In preparation for flow cytometric analysis of CSF3R expression, up to five million cells were
added to a 5 mL FACS tube (Becton, Dickinson and Company, Franklin Lakes, NJ) and centrifuged at 300
g at 4C for 5 minutes. Cells were next incubated with Fc Block (clone 2.4G2, Becton, Dickinson and
Company, Franklin Lakes, NJ) and PE-conjugated anti-CD114 antibody (clone LMM741, Becton,
Dickinson and Company, Franklin Lakes, NJ) at a 1:100 dilution in 100 µL of 1X FACS buffer for 1 hour on
ice.
SILAC cell culture and heavy amino acids labeling
The virally transduced BaF3 cell lines were grown in RPMI medium (Invitrogen, Carlsbad, CA)
supplemented with 5% fetal bovine serum (FBS), 2mM L-glutamine, 100 U/mL penicillin and 100 ug/mL
Pencillin/streptomycin in a incubator at 37 ?C with 5.0% CO2. The cells were maintained in RPMI without
lysine and arginine supplemented with 5% FBS, 2mM L-glutamine, 100 U/mL penicillin and 100 ug/mL
streptomycin, 50 mg/L arginine-12C6 monohydrochloride and 100 mg/L lysine-12C6 monohydrochloride
(light) or 50 mg/L arginine-13C6 monohydrochloride and 100 mg/L lysine-13C6 monohydrochloride (heavy)
(Cambridge Isotope Laboratories) for at least 5 doublings (about 6 days) to ensure uniform incorporation
of the heavy amino acids for SILAC labeling purposes. Heavy isotope incorporation from a small aliquot
of cells was verified to be greater than 95% by mass spectrometry. Upon verification of labeling
efficiency 100 million cells in an exponential growth phase were washed 3 times with PBS before 6 hours
of serum starvation prior to G-CSF stimulation at various time points. Cells grown in heavy SILAC
medium were stimulated with G-CSF (40 ng/mL) for 12.5 mins and 90 mins at 37 ?C and cells grown in
light medium were left unstimulated.
Cell lysis and protein digestion on SILAC samples
G-CSFR stimulated or unstimulated SILAC labeled BaF3 cells were washed with cold PBS twice
and lysed in lysis buffer (20 mM HEPES pH 8.0, 9 M urea, 1 mM sodium orthovanadate, 2.5 mM sodium
pyrophosphate, 1 mM beta-glycerophosphate), sonicated and centrifuged at 20,000 x g at 4?C for 30
min. Post-stimulation, the total protein amount was determined using 660 nm assay (Thermo Scientific,
#22660). Equal amounts of protein (10 mg from light and 10 mg from heavy labeled cells) were mixed,
before reduction and alkylation steps. The mixed lysates were reduced using DTT at a final
concentration of 5 mM at 60?C for 20 min and alkylated using 10 mM iodoacetamide for 10 min at RT in
the dark. The samples were diluted so that final urea concentration was <2M with 20 mM HEPES, pH 8.0 and further digested with TPCK treated trypsin (Worthington Biochemical Corp) overnight at room temperature with rotation. Subsequently, the trypsin digested peptides were acidified using 1% Triflouroacetic acid (TFA) and further desalted using C-18 Sep-pak cartridge (Waters, cat# WAT051910). Lyophilization was performed on the peptides at least for 3 days before phospho-tyrosine enrichment. Immunoaffinity purification of tyrosine phosphopeptides Immunoaffinity purification (IAP) of phosphopeptides was carried out as described earlier (2, 3). In summary, 20 mg of the lyophilized peptide mixture was dissolved in 1.4 ml of IAP buffer (50 mM MOPS pH 7.2, 10 mM sodium phosphate, 50 mM NaCl) with the pH of 7.2. Prior to IAP procedure, the PTyr-1000 beads (Cell Signaling Technology, #8803) were washed with IAP buffer at least three times at 4?C. The incubation of the peptide mixture with P-Tyr-1000 beads was done for 30 min with gentle rotation. The beads were washed three times with ice cold IAP buffer following additional two washes with ice cold HPLC grade water. Peptides elution was performed with 50 µL of 0.15% TFA twice at room temperature then desalted using C-18 Stage Tips columns (3). Nano-LC-MS/MS Analysis A TripleTOF 5600+ MS (Sciex, Concord, ON, Canada) coupled to an Eksigent (Dublin, CA) nanoLC Ultra nanoflow system was used to run nano-LC-MS/MS analyses. Formic acid (0.1%) was used to reconstitute the dried phosphopeptide samples before loading onto IntegraFrit trap column (outer diameter of 360 µm, inner diameter of 100 µm, and 25 µm packed bed) from New Objective (Woburn, MA) at 2 µL/min in formic acid /water 0.1/99.9 (v/v) for 10 min to concentrate the sample. Furthermore, the trap column was switched to align with the analytical column for chromatographic separation of the sample. The analytical column used was Acclaim PepMap100 (inner diameter of 75 µm, length 15 cm, C18 particle size of 3 µm, and pore size of 100 A) from Dionex-Thermo Fisher Scientific (Sunnyvale, CA). The phospho-peptide elution was performed using a varying mobile phase (MP) gradient from 95% phase A (0.1% FA in water to 40% phase B (0.1% FA in acetonitrile) for 70 min, from 40% phase B to 85% phase B for 5 min, and then keeping the same MP composition for 5 more minutes at 300 nL/min. The method used to separate the peptides from mass spectrometer was operated in positive ion mode using 4303 cycles for 90 min, where each cycle consisted of one TOF-MS scan (0.25 s accumulation time, in a 350 to 1600 m/z window), following 20 information dependent acquisition (IDA) MS/MS-scans on the most intense candidate ions, with minimum of 150 counts. Product ion scan was operated under vender specified high sensitivity mode (an accumulation time of 0.05 s and a mass tolerance of 100 ppm). Mass spectrometric data analysis The nano-LC-MS/MS generated data files (.wiff files) containing the enriched phosphopeptides were further analyzed for protein identification and quantification using Protein PilotTM software (version 5.0, revision 4769) which integrates the Paragon algorithm, searched against a UniProt database of Mus musculus protein sequences. Individual phospho-tyrosine enriched raw MS data was processed using the SILAC settings in Protein Pilot with minor changes: sample type (SILAC (Lys+8, Arg+10)), Cys Alkylation (Iodoacetamide), Digestion (Trypsin), Instrument (Triple TOF 5600), Special Factors (Phos-Tyr affinity column). False Discovery Rate (FDR) was set at 0.05 with through ID as search effort. The searched .group files were used to export a spreadsheet as containing the complete peptide summary report. Only those phosphopeptides identified with a minimum of 95% confidence (calculated by probability algorithm of Protein Pilot software) were included in the relative quantitation and subsequent data analyses. Unique phosphopeptides were then selected based on sequence, modification, mass-to-charge ratio (m/z value), and charge (z) as previously published by Wijeratne et al. (4). A heavy/light ratio for each phosphopeptide was calculated based on MS1 quantification values generated by Protein Pilot. Data Analysis and Bioinformatics In order to pre-process and normalize the phospho-proteomic data set, we created a custom Perl script which took the raw data (one file per replicate of an experimental condition) and produced a table of median-normalized ratios of peptide intensities for receptor-activated vs. non-activated states (G-CSF treated vs. non-G-CSF treated). Peptide identifications were filtered for 95% confidence, intensities of multiple identifications of the same peptide are averaged. Special consideration was given to cases where a peptide was detected in only one of the two channels (activated or non-activated receptors), in which log ratios were artificially set to plus or minus infinity. Ratios were normalized within replicates to have 0 median. Technical reproducibility was assessed across replicates and plotted with Pearson correlations at >0.80. Due to the degree of reproducibility, missing values were imputed
to that of the replicate (if available), otherwise missing values were imputed to the median value of 0
(no change between activated and non-activated states) before final analysis. Perl processed data was
further used for heat map and unbiased clustering analyses in using R: A language and environment for
statistical computing (http://www.R-project.org) (5). STRING db software (6) was used to determine the
interaction network of identified proteins showing differential phosphorylation changes. The
identification of these interactions was determined by neighborhood in the gene, gene fusion, cooccurrence
across genomes, co-expression, experimental data, databases, text mining and homology.
For simplicity of visualization purposes, the network was manually curated showing only the direct
interaction between two proteins. Kinase Enrichment Analysis (KEA) was used to identify upstream
kinases regulating phosphorylation pattern (7). KEA is an online web-based bioinformatics tool which
utilizes a database of mammalian proteins/genes and their respective potential kinases. The
computation of probable kinase is performed based on distribution of kinase-substrate proportions in
comparison with distribution of kinase-substrate in the database and its association with an input
protein list with the specific phosphorylation sites (7).
G-CSF induction and immunoblotting
BaF3 cell expressing normal and mutated G-CSFRs were serum starved for 6 hours before the
induction with 40 ng/mL of G-CSF. The induced cells were washed 1 time with ice cold PBS before lysis.
The cells were lysed in protein extraction buffer (20 mM Tris-HCl, 150 mM Sodium Chloride, 2 mM EDTA,
1 mM EGTA, Complete Mini Protease Inhibitor Cocktail Tablet (Roche), 10 mM Sodium fluoride, 1 mM
Sodium orthovanadate, 1 mM beta-glycerophosphate, 1% NP-40, 1% Tween-20, 10% Glycerol, 2.5 mM
Sodium pyrophosphate, 1 mM PMSF). Lysates were sonicated at 15 W for 3 pulses of 10 sec each with a
5 second interval in between pulses on ice. After lysing, cells were centrifuged at 20,000 g for 10 min at
4C. The supernatant was collected and protein estimation was performed using 660 nm assay (Thermo
Scientific) (#22660). 20-25 µg of total lysate was used for each immunoblot. The protein separation was
performed using a 4-12 % gradient SDS-PAGE gel. The protein transfer was performed on PVDF
membrane using a semi-dry apparatus running at 15V for 30 min. The membrane was blocked with 5%
milk solution in TBS-T (0.1% Tween-20, Invitrogen) before primary antibody probing. Antibodies were
purchased from Cell Signaling Technology: Phospho-Stat5 (Tyr 694) (C11C5) (#9359), Phospho-Stat3
(Tyr705) (D3A7) (#9145), Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) (#4370), Stat5
(#9363), Stat3 (#4904), p44/42 MAPK (Erk1/2) (#9102), Phospho-BTK (Tyr223) (D9T6H) (#87141), BTK
(#8547), Phospho-Akt (Ser473) (D9E) (#4060), Akt (#9272), Actin (#4970). The dilution of the primary
antibody used was per manufacturer’s guidelines. Anti-rabbit and anti-mouse secondary antibodies
conjugated with HRP (GE Healthcare) were used at a 1:5000 dilution 5% Milk in TBS-T. All immunoblots
were developed on ChemicDocTM touch imaging system (Bio-Rad). Every immunoblot was replicated at
least twice.
Bone Marrow Stem/Progenitor Isolation, cell proliferation and viability experiments
Bone marrow cells were isolated from freshly sacrificed mice by crushing bones using a mortar
and pestle in 1X FACS Buffer (1% FBS, 0.01% NaN3 in DPBS) under sterile conditions and passed through
a 40 µm cell strainer (Becton, Dickinson and Company, Franklin Lakes, NJ) to obtain single cell
suspensions for downstream applications. To enrich for stem/progenitor populations, freshly isolated
cells were incubated with CD117 MicroBeads and separated on an AutoMACS Pro separator (Miltenyi)
according to manufacturer specifications. 20,000 32D or c-Kit enriched cells were seeded in 96 well plate
in 200 µl of RPMI medium in an increasing concentration of Ibrutinib. Ibrutinib concentrations used
were 5 nM, 10 nM, 25 nM, 50 nM, 100 nM, 250 nM, 500 nM, 750 nM, 1000 nM, 2500 nM, 5000 nM in a
final concentration of 0.1% DMSO for 32D cells and 1 nM, 10 nM, 100 nM, 250 nM, 1000 nM in a final
concentration of 0.1%DMSO for c-Kit cells. After treatment, cells were incubated for 24 h at 37C, 5%
CO2. . Cell proliferation and viability was determined using Trypan Blue live/dead hemocytometer cell
counting. 

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