Supplementary Figure 1: XMAP215 stimulates microtubule nucleation in Xenopus egg extracts, Related to Fig. 1.

(A) Addition of excess wild-type XMAP215-GFP to Xenopus egg extracts in the presence of 5.5 μM RanQ69L. Representative images are displayed at 300 seconds of the reaction. Scale bar, 10 μm. See Supplementary Video 1. The experiment was repeated three times with independent extract preparations, with more than four additional supporting experiments. (B) EB1 comets in the entire field of view were counted as readout of microtubule (MT) nucleation. The number of MTs was plotted over time. The analysis was performed thrice with independent extract preparations. (C) Growth speed of MTs was obtained by tracking the EB1 comets over the image sequence. The number of growth speed measurements obtained from consecutive frames of all tracks (n): buffer (n = 17,798), 120 nM (n = 62,633), 240 nM (n = 75,532), 480 nM (n = 109,138). Mean growth speed (diamonds) is almost constant at 10 μm/min. Error bars display s.d. in growth speed across all measurements. The analysis was performed twice on experiments performed with independent extract preparations. (D) EB1 comets in the field of view were detected, counted and plotted over time for reactions from Fig. 1B. The nucleation measurements were repeated more than four times for experiments performed with independent extract preparations. Wild-type XMAP215-GFP was added back at 85 nM (equivalent to 120 nM in IgG-depleted extracts). MT lifetime (mean ± s.d.) was estimated to be 35 ± 15 seconds (IgG depletion), 43 ± 41 seconds (XMAP215 depletion + wt XMAP215). Lifetime of MTs was defined as the time from appearance of EB1 comet on the plus end (nucleation) to first disappearance of the EB1 comet from the plus end (catastrophe or pause) for n = 7 MTs analyzed. MT lifetime measurements were performed manually on one representative experiment displayed in Fig. 1B. (E) Related to Fig. 1A-B. Western blot analysis of α-tubulin, TACC3, TPX2, augmin subunits (HAUS1 and HAUS6), γ-TuRC subunits (γ-tubulin and GCP4) in XMAP215 immunodepletion, confirming that none of these proteins are co-depleted with XMAP215. The experiment was repeated identically at least twice with independent extracts and immunodepletions, and subsets of all proteins displayed were immunoblotted in more than three additional independent experiments. See Supplementary Fig. 9 for unprocessed scans.