Figure 6

(a) Immunofluorescent staining of activated FAK in SH-SY5Y cells. SH-SY5Y cells were plated on laminin coated coverslips in complete DMEM (undifferentiated) or differentiated in serum free DMEM with IGF-1 for 72 hours. Cells were fixed in 4% paraformaldehyde, permeabilised in PHEM/0.1% Triton X and blocked in PHEM/5% goat serum. Cells were stained with either pFAK Y³⁹⁷ or pFAK Y⁹²⁵ (red) and the nuclei were stained with Hoechst 33242 (blue). All images were acquired sequentially at 63× and images merged using ImageJ. Scale bar = 20 μm. (b) FAK was immunoprecipitated from lysates of SH-SY5Y cells, undifferentiated or differentiated with IGF-1 and run on 12% SDS-PAGE gels and probed for phospho-tyrosine and FAK, followed by detection with LI-COR Odyssey™. Densitometry of protein bands was measured using LI-COR Odyssey™ software and normalised to undifferentiated levels. The fold increase in pFAK signal compared to undifferentiated protein level are plotted on a bar chart ± SEM, n = 3.