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Comprehensive Analysis of Interactions between the Src-Associated Protein in Mitosis of 68 kDa and the Human Src-Homology 3 Proteome

Figure 1

Analysis of Sam68-binding SH3 domains in vitro.

(A) Phage-ELISA analysis: Recombinant Sam68 was coated in 96-well-plates and incubated with dilution series of individual SH3-phage supernatants. The amounts of bound phages were measured using a horseradish peroxidase-conjugated anti-M13-specific antibody with TMB-substrate detection at 450 nm (mean of three independent experiments, normalized to maximum OD-values). (B) Apparent Kd-values for Sam68-SH3-phage-interactions were derived from the corresponding Scatchard-Plots (suppl. Fig. S2) to the data from (A), accounting for correction of SH3 domain concentrations. (C) Pull-down assay: Purified GST-SH3 domains were immobilized on magnetic beads and incubated with an MT-4 cell lysate. Bound proteins were eluted and Sam68 was detected by western blot analysis.

Figure 1

doi: https://doi.org/10.1371/journal.pone.0038540.g001