The membrane was blocked for at least 2?h in 2% BSA in TBST (10?mM Tris (pH 7.5), 150?mM NaCl, 0.05% Tween 20) before incubation with primary antibody overnight at 4?C and with secondary antibody for 1?h at room temperature. Y1422/Y1440, but were unable to verify this conversation in A431 carcinoma Monotropein cells that overexpress the EGFR. Furthermore, using A431 cells devoid of 4 or reconstituted with phenylalanine specific mutants of 4, the activation of several downstream signaling pathways, including PLC/PKC, MAPK and PI3K/Akt, were not substantially affected. We conclude that tyrosine-phosphorylated 4 does not enhance EGFR-mediated signaling in EGFR-overexpressing cells, despite the fact that this integrin subunit is usually highly tyrosine phosphorylated in these cells. for 60?min at 4?C and supplemented Igfbp6 with SDS sample buffer (50?mM TrisCHCl pH 6.8, 2% SDS, 10% glycerol, 12.5?mM EDTA, 0.02% Bromophenol Blue) with -mercaptoethanol and heated at 95?C for 5 min63. Immunoprecipitations were performed with the cleared cell lysates after centrifugation. The lysates were incubated at 4?C for 1.5C2?h with 1?g antibody. Subsequently, the lysates were incubated 4C20?h at 4?C with prewashed Protein G Sepharose 4 Fast Flow beads (GE Healthcare), beads were washed Monotropein two times with lysis buffer and two times with PBS and bound proteins were dissolved in SDS sample buffer with -mercaptoethanol and heated at 95?C for 5?min. Proteins were separated on 4C12% Bolt gradient gels (Invitrogen) and transferred to Immobilon-P transfer membranes (Millipore). The membrane was blocked for at least 2?h in 2% BSA in TBST (10?mM Tris (pH 7.5), 150?mM NaCl, 0.05% Tween 20) before incubation with primary antibody overnight at 4?C and with secondary antibody for 1?h at room temperature. After each incubation step, the membranes were washed twice with TBST and twice with TBS (TBST without Tween 20) as described previously64. Antibodies were detected using Clarity Western ECL Substrate (Bio-Rad). Quantification of western blot data was performed with ImageJ. RT-PCR Reverse transcriptaseCquantitative polymerase chain reaction (RT-qPCR) analysis was performed as described previously64. Reactions were performed in triplo for determination of the levels of Yes, Lyn, Frk, Lck, Fgr, Hck, Blk and CyclofilinA (CF; control) in A431 cells. Cells were grown in complete medium and lysed in RNA-Bee (Tel-test Inc.) or Trizol reagent (Ambion #15596018), following manufacturers instructions. Total RNA was separated from DNA and proteins by the addition of chloroform (Sigma-Aldrich) and subsequent centrifugation at 12,000??for 15?min at 4?C. The RNA was precipitated with isopropanol and washed with 70% ethanol. Integrity of the isolated RNA was assessed by agarose gel electrophoresis. First strand cDNA synthesis was performed with 3?g of total RNA using the first strand cDNA synthesis kit K1612 (Thermo Fisher Scientific) following manufacturers directions. The PCR reactions were run using SYBR Advantage qPCR premix (Clontech) on a 7500 Fast Real-Time PCR system (Applied Biosystems) and the primers (IDT) which were used, are shown in Table S2. Relative mRNA quantities were obtained using the 2 2?Ct method. The Ct is the obtained Ct value of the SFK minus the obtained Ct value of CF (endogenous control). Peptide pulldowns and mass spectrometry analyses Phosphorylated and unphosphorylated peptides based on sequences within the integrin 4 (Table S3) were synthesized by Fmoc chemistry in the laboratory of Huib Ovaa at the NKI and purified by HPLC. The peptides were synthesized with a cysteine followed by a caproic acid at the amino terminus. The cysteine was used for coupling to SulfoLink (Pierce, Rockford, IL), while the caproic acid was added to provide spacing between the peptide and the support material. The sequence of each peptide was verified by mass spectrometry. For peptide pull downs, PA-JEB/4 keratinocytes were produced to confluency on 15?cm tissue culture dishes, lysed in 3?ml 50?mM HEPES pH?7.5, 150?mM NaCl, 10% glycerol, 1% Triton X-100, 1.5?mM MgCl2, 1?mM EGTA, 100?mM NaF, 10?mM sodium pyrophosphate, 500?M sodium vanadate, 10?g?ml?1 aprotinin and 10?g?ml?1 leupeptin (PLC-lysis buffer) per 15?cm tissue culture dish. Lysates were precleared by centrifugation at 10,000?rpm in a Monotropein microfuge at 4?C for 30?min. and incubated for 1?h with 100?l of a 10% slurry of each of the different 4 peptides. Agarose beads were collected by centrifugation and washed four times with PLC buffer. Sample were boiled, resolved by SDS-PAGE and stained with Coomassie blue. Protein bands of interest were cut out, subjected to in-gel trypsin digest and analyzed by mass spectrometry. Monotropein Proliferation assay Cells were seeded in 96 well plates at a density of 5000 cells per well. Cells were collected for 4 consecutive days. The cells were washed with PBS, fixed with 2% paraformaldehyde for 10?min, washed with PBS and stained with 2% crystal violet for 10?min. After washing with demiwater, plates were dried overnight and cells were lysed in 2% SDS. Absorbance was measured at 590?nm on an Epoch microplate spectrophotometer (BioTek). Supplementary information Supplementary Informations.(45M, pdf) Acknowledgements We thank Monique.