Fig. 2

Sharpin directly binds to the integrin β1 CT and inhibits the talin head domain binding. a Purified GST and GST-fused integrin CT, as indicated, were coupled to Glutathione Sepharose beads and used to incubate with his-tagged sharpin (His-SH). After incubation, the beads were extensively washed and proteins bound to the beads were eluted by boiling the beads in laemmli sample buffer. GST proteins loaded on the beads and co-precipitated His-SH were evaluated by SDS-PAGE followed by Coomassie blue (C. blue) staining and immunoblotting (IB). b The N-terminus (SH-N, 1–217 amino acids) and C-terminus (SH-C, 217–387 amino acids) of sharpin were expressed and purified with a his tag and used to test their binding to GST or GST-β1 CT, as described in (a). c Selected region of HSQC spectra of 50 μM ¹⁵N-labeled β1 CT in the absence (black) and presence (red) of 250 μM N-terminus of sharpin (SH-N). d Selected region of HSQC spectra of 50 μM ¹⁵N-labeled β3 CT in the absence (black) and presence (red) of 250 μM N-terminus of sharpin (SH-N). e, f Purified protein of the N-terminus of sharpin was immobilized on CM5 chip surfaces. Various concentrations (2.5 μM, 5 μM, 10 μM, 20 μM and 40 μM) of either the integrin β1 CT protein (e) or the integrin β3 CT protein (f) were injected and passed over the chips, and the binding curves were recorded on a Biacore 8 K instrument. g Purified GST and GST-β1 CT proteins were loaded onto Glutathione Sepharose beads which were then used for incubating with flag-fused talin head (Flag-TH) in the presence or absence of his-sharpin (His-SH) with different ratios. After incubation, the beads were washed and co-precipitated proteins were measured by SDS-PAGE followed by Coomassie blue (C. blue) staining and immunoblotting (IB). h Purified GST, GST-β1 CT and GST-β1 CT mutants that carry the NPIY/AAAA mutations or the KSAV/AAAA mutations were coupled to Glutathione Sepharose beads and used to incubate with His-SH, Flag-TH or kindlin-1 (K1) proteins, respectively. Binding of His-SH, Flag-TH or K1 to these GST proteins were evaluated by SDS-PAGE followed by immunoblotting (IB). Meanwhile, the loaded GST proteins on the beads were also measured by Coomassie blue (C. blue) staining