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. Author manuscript; available in PMC: 2014 Dec 12.
Published in final edited form as: Mol Cell. 2013 Oct 31;52(5):10.1016/j.molcel.2013.10.002. doi: 10.1016/j.molcel.2013.10.002

Figure 4. The HBIS directly inhibits xHaspin.

Figure 4

(A) Gel code blue staining of purified MBP-xHaspin-ΔN729 (1) and MBP-xHaspin-ΔN729ΔHBIS (2).

(B) Signal intensity of Western blots using the anti-H3T3ph antibody (A.U.) was plotted against the amount of H3T3ph (loaded). The signal intensity showed a linear relationship to the input H3T3ph.

(C) Initial velocities of H3T3 phosphorylation reaction using 1 nM MBP-xHaspin-ΔN729 or MBP-xHaspin-ΔN729ΔHBIS at various H31–45-GST concentration are shown. Triplicate data sets were fit to the Michaelis-Menten equation using the nonlinear least square fit method. Error bars represent standard error of the mean.

(D) Km and kcat values were obtained after fitting the data in C to the Michaelis-Menten equation.

(E) Either HBIS- or SCR-peptides were added at the indicated concentration to the in vitro kinase reaction with 1 nM xHaspin-ΔN729 and 200 nM H31–45-GST. Substrate phosphorylation was detected by Western blots using anti-H3T3ph antibody.

See also Figure S4.