Computational protocol: Phosphoproteomics of collagen receptor networks reveals SHP-2 phosphorylation downstream of wild-type DDR2 and its lung cancer mutants

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Protocol publication

[…] HEK-293-DDR2 cells were grown in six-well plates for 24 h. Cells were then dissociated with non-enzymatic cell dissociation solution (Sigma) and resuspended in PBS containing 1% BSA. The cells were incubated for 30 min on ice with primary mAbs (monoclonal antibodies) at 10 μg/ml in 100 μl of PBS/BSA. Cells were then washed three times with PBS/BSA and incubated with FITC-conjugated goat anti-mouse IgG (F-9006, Sigma) for 30 min on ice. After three washes as above, the cells were resuspended in 2% formaldehyde in PBS. Data were subsequently collected on a BD LSRFortessa cell analyser using BD FACSDiva software 6.0 (BD Biosciences), and further analysed on FlowJo software 7.6.4 (Tree Star). Mouse anti-(integrin α1) mAb, clone FB12, was purchased from Millipore Chemicon and mouse anti-(integrin α2) mAb, clone AK7, was from AbD Serotec. Mouse anti-(integrin β1), clone P5D2, was purified from hybridoma cells obtained from the Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA, U.S.A. […]

Pipeline specifications

Software tools BD FACSDiva, FlowJo
Application Flow cytometry
Diseases Carcinoma, Squamous Cell, Lung Neoplasms
Chemicals Tyrosine