P tyr 100 phosphotyrosine antibody beads

Protein concentrations were measured using the BCA Protein Assay (Thermo
Fisher Scientific). Reduction, alkylation, and trypsin digestion were performed
as previously described (32 (link)). For
phosphotyrosine peptide enrichment, peptide immunoprecipitation was performed
using p-Tyr-100 phosphotyrosine antibody beads (Cell Signaling Technology) as
previously described (32 (link)). Prior to
immunoprecipitation, a 5 pmol fraction of synthetic phosphopeptide LIEDAEpYTAK
was added to each individual replicate and time point sample as a normalization
standard to correct for any variation in mass spectrometer sensitivity between
individual replicates. Samples were then desalted using ZipTip pipette tips (EMD
Millipore, Billerica, MA) according to the manufacturer’s
instructions.

To halt the culture, media was removed from the cells and lysis buffer (9 M urea, 1 mM sodium orthovanadate, 20 mM HEPES, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, pH 8.0) was added to the cells. Lysates were prepared from the co-culture of CAF and SKOV3ip1 for 4 hours or the control cells with lysates of CAF alone and SKOV3ip1 alone mixed together. Lysates were sonicated at a 30 watt output with 2 bursts of 30 seconds each and cleared at 20,000×g for 15 minutes at 4°C. Lysates were reduced with 45 mM DTT for 20 minutes at 60°C, and alkylated with 100 mM iodoacetamide for 15 minutes at room temperature in the dark. Lysates were then diluted 4-fold with 20 mM HEPES buffer, pH 8.0 and digested with TPCK-treated trypsin (Worthington, Lakewood, NJ) in a 1:1 (w/w) trypsin:protein ratio overnight at room temperature. Tryptic peptides were acidified to pH 2.0 with 20% trifluoroacetic acid (TFA), cleared at 1,800×g for 5 minutes at room temperature, and desalted using C18 Sep-Pak plus cartridges (Waters, Milford, MA). Eluents containing peptides were lyophilized for 48 hours to dryness. Peptide immunoprecipitation was performed using p-Tyr-100 phosphotyrosine antibody beads (Cell Signaling Technology) as previously described (Helou et al., 2013 (link)).