Quantitative analysis of neurite outgrowth behaviour on different substrates. (a) SH-SY5Y cells were plated on uncoated (plastic) or surfaces coated with 10 μg/ml of different ECM proteins: laminin, collagen or fibronectin. Cells were incubated in regular DMEM media containing 10% FBS for 24, 48 or 72 hours. Cells were counted from each condition at each timepoint and the number of differentiated cells was expressed as a percentage of the total cells counted ± SEM, n = 3. (b) The length of the neurites extending from the SH-SY5Y cells after 72 hours differentiation were measured and the average length for each matrix was expressed in a graph ± SEM, n = 3. Significant differences were measured by ANOVA (#P < 0.05 for comparisons between ECM protein-coated substrates and plastic; *P < 0.05 for comparisons between the coated substrates, Collagen vs. Laminin, Laminin vs Fibronectin or Collagen vs. Fibronectin). (c) Cells were lysed at each timepoint and run on 12% SDS-PAGE gels and probed for focal adhesion kinase (FAK), followed by detection with LI-COR Odyssey™, to monitor effect of matrices and time on protein levels. (d) To monitor the effect of the matrices on total FAK phosphorylation, cells were grown on each of the matrices for 24 hours before lysing. FAK was immunoprecipitated and the immunoprecipitate was run on 12% SDS-PAGE gel and probed for phospho-tyrosine and FAK followed by detection using the LI-COR Odyssey™ infrared image scanner.