Figure 2
PDI binds VWF. (A) Coimmunoprecipitation of endogenous PDI and VWF from HUVEC lysates. Immunoprecipitation was performed using either rabbit anti-VWF antibody or control rabbit IgG bound to protein G-agarose. Coimmunoprecipitating proteins were separated by sodium dodecyl sulfate gel electrophoresis, and PDI was detected using mouse anti-PDI. Lanes: input, cell lysate of untreated HUVECs; VWF, immunoprecipitate from HUVEC lysate employing anti-VWF antibody; control, immunoprecipitate from HUVEC lysate employing rabbit control IgG. (B-D) MST analyzes the movement of molecules in a temperature gradient (B). (C) Binding of PDI to VWF was detected as a decrease in thermophoretic depletion. (D) PDI binds to wtVWF (black triangles), the isolated CK domain (gray squares), and p.Cys2771Arg (gray circles) with a very similar affinity (KD = 240 ± 100 nM). (E-G) FCS detects the diffusive dynamics of molecules (E). (F) Binding results in prolonged diffusion times visualized by a shift of the autocorrelation curve G(τ). (G) In agreement with the MST measurements, we detect similar affinities for wtVWF (black triangles) and mutant p.Cys2771Arg (gray circles) (KD = 300 ± 100 nM).

PDI binds VWF. (A) Coimmunoprecipitation of endogenous PDI and VWF from HUVEC lysates. Immunoprecipitation was performed using either rabbit anti-VWF antibody or control rabbit IgG bound to protein G-agarose. Coimmunoprecipitating proteins were separated by sodium dodecyl sulfate gel electrophoresis, and PDI was detected using mouse anti-PDI. Lanes: input, cell lysate of untreated HUVECs; VWF, immunoprecipitate from HUVEC lysate employing anti-VWF antibody; control, immunoprecipitate from HUVEC lysate employing rabbit control IgG. (B-D) MST analyzes the movement of molecules in a temperature gradient (B). (C) Binding of PDI to VWF was detected as a decrease in thermophoretic depletion. (D) PDI binds to wtVWF (black triangles), the isolated CK domain (gray squares), and p.Cys2771Arg (gray circles) with a very similar affinity (KD = 240 ± 100 nM). (E-G) FCS detects the diffusive dynamics of molecules (E). (F) Binding results in prolonged diffusion times visualized by a shift of the autocorrelation curve G(τ). (G) In agreement with the MST measurements, we detect similar affinities for wtVWF (black triangles) and mutant p.Cys2771Arg (gray circles) (KD = 300 ± 100 nM).

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