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Figure 6

$(A) Akirin2–histone H3.1 interaction by recAkirin2 binding to a histone peptide array, which resulted in the identification of histone H3 105–124. Relative fluorescence is shown in RFU. (B) Corroboration of the interactions between recAkirin2 (produced in insect cells and E. coli) and histone H3 105–124 by in vitro protein pull-down and Western blot with Akirin2-specific primary antibodies. Immunoreactive proteins were visualized by chemiluminescence. Streptavidin beads incubated only with Akirin2 and recAkirin2 were included as negative (C–) and positive (C+) controls, respectively. (C) Corroboration of recAkirin2–histone H3 105–124 interactions by ITC. Calorimetric titrations of histone H3 105–124 into Akirin2 (left) and BSA (right). Upper plots show the thermogram and lower plots show the interaction isotherm. Non-linear regression data analysis allowed estimating the interaction parameters for Akirin2 (continuous red line): Ka, association constant; Kd, dissociation constant; ΔH, interaction enthalpy; n, fraction of active (binding-competent protein). No interaction was observed for BSA.$

Characterization of Akirin2–histone H3 105–124 physical interactions

(A) Akirin2–histone H3.1 interaction by recAkirin2 binding to a histone peptide array, which resulted in the identification of histone H3 105–124. Relative fluorescence is shown in RFU. (B) Corroboration of the interactions between recAkirin2 (produced in insect cells and E. coli) and histone H3 105–124 by in vitro protein pull-down and Western blot with Akirin2-specific primary antibodies. Immunoreactive proteins were visualized by chemiluminescence. Streptavidin beads incubated only with Akirin2 and recAkirin2 were included as negative (C–) and positive (C+) controls, respectively. (C) Corroboration of recAkirin2–histone H3 105–124 interactions by ITC. Calorimetric titrations of histone H3 105–124 into Akirin2 (left) and BSA (right). Upper plots show the thermogram and lower plots show the interaction isotherm. Non-linear regression data analysis allowed estimating the interaction parameters for Akirin2 (continuous red line): K_a, association constant; K_d, dissociation constant; ΔH, interaction enthalpy; n, fraction of active (binding-competent protein). No interaction was observed for BSA.

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