Supplementary Figure 5: Microtubule nucleation by XMAP215 protein constructs, Related to Fig. 5. | Nature Cell Biology

Supplementary Figure 5: Microtubule nucleation by XMAP215 protein constructs, Related to Fig. 5.

From: XMAP215 is a microtubule nucleation factor that functions synergistically with the γ-tubulin ring complex

Supplementary Figure 5

(A) Branching MT nucleation activity of TOG3-CT construct of XMAP215, added back to XMAP215-depleted extracts at 120 nM final concentration in the presence of 5.5 μM RanQ69L and 1 μM GST-TPX2 α3-7. Representative image is displayed at 560 seconds of the reaction. Scale bar, 10 μm. The experiment was repeated three times with independent extract preparations. See Supplementary Video 6. (B) EB1 comets in the entire field of view were counted and plotted with time for addition of individual N-terminal deletion constructs of XMAP215. The analyses were repeated twice on experiments performed with independent extract preparations. See Fig. 5a. (C-D) Branching MT nucleation activity of full-length XMAP215 1-5AA construct was compared with wild-type XMAP215. All proteins were added back to XMAP215-depleted extracts at 120 nM concentration in the presence of 5.5 μM RanQ69L and 1 μM GST-TPX2 α3-7. Full-length protein with mutant TOG domains did not support any MT nucleation. Representative images are displayed at 722.5 seconds of the reaction. The number of EB1 comets was quantified as in (B). The experiment was repeated thrice with independent extract preparations, and the analysis was repeated on two independent experimental sets.

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