Figure 5. An N-terminal fragment of... | Rockefeller University Press

Figure 5.

$Figure 5. An N-terminal fragment of CENP-F binds better to curls than MTs, whereas a C-terminal segment does the opposite. (A) 0.2 µM N558 or 0.4 µM 2592C were mixed with buffer alone or with buffer including 0.4 µM tubulin assembled as GTP curls, GDP curls, or Tx-MTs, and then incubated and centrifuged as described in Materials and methods. Gels of supernatant and pellet fractions were stained with SYPRO Ruby (Molecular Probes). (B) Bound protein was measured by densitometry of the pellet fractions. The fraction of protein that pelleted nonspecifically in the buffer-only lane was subtracted from the values measured for each experiment. The amounts shown are from a single representative experiment out of four repeats for N558 and two repeats for 2592C. (C) 0.2 µM N558 was mixed with various concentrations of tubulin added as rings, curls, or Tx-MTs, and then incubated, centrifuged, and analyzed by gel electrophoresis and densitometry, as described in Materials and methods. (D) Protein amounts in each lane of three to five experiments, as in C, were quantified as in Materials and methods and the results were plotted. The data were fit with a quadratic model for protein binding by the method of least squares. Normalized score functions for goodness of each fit are shown in the bottom row. A value of 0 for this function would imply a perfect fit.$

An N-terminal fragment of CENP-F binds better to curls than MTs, whereas a C-terminal segment does the opposite. (A) 0.2 µM N558 or 0.4 µM 2592C were mixed with buffer alone or with buffer including 0.4 µM tubulin assembled as GTP curls, GDP curls, or Tx-MTs, and then incubated and centrifuged as described in Materials and methods. Gels of supernatant and pellet fractions were stained with SYPRO Ruby (Molecular Probes). (B) Bound protein was measured by densitometry of the pellet fractions. The fraction of protein that pelleted nonspecifically in the buffer-only lane was subtracted from the values measured for each experiment. The amounts shown are from a single representative experiment out of four repeats for N558 and two repeats for 2592C. (C) 0.2 µM N558 was mixed with various concentrations of tubulin added as rings, curls, or Tx-MTs, and then incubated, centrifuged, and analyzed by gel electrophoresis and densitometry, as described in Materials and methods. (D) Protein amounts in each lane of three to five experiments, as in C, were quantified as in Materials and methods and the results were plotted. The data were fit with a quadratic model for protein binding by the method of least squares. Normalized score functions for goodness of each fit are shown in the bottom row. A value of 0 for this function would imply a perfect fit.