Supplementary Figure 4: Characterization of purified γ-TuRC, Related to Fig. 4.

(A) Isolation of γ-TuRC from Xenopus egg extracts. Mass spectrometric analysis of prominent bands in the elution showed all γ-TuRC components: γ-tubulin, GCPs 2–6, Mzt2b and NEDD1. Some contaminating factors: PCM1, Clathrin heavy chain, SPAG5 and small kinetochore associated protein (KNSTRN) were still present. Representative image is displayed, and mass spectrometry was repeated twice with similar results. (B) Input, flow-through, and elution from the affinity purification were analyzed by immunoblot for XMAP215, TACC3, α-tubulin and TPX2. These components were not found in the elution, while γ-TuRC specific factors (γ-tubulin, GCP4, NEDD1) were present in purified γ-TuRC. These findings were consistent when repeated with two independent γ-TuRC preparations. (C) Purified γ-TuRC and XMAP215 were incubated to promote MT nucleation. For 5 nM XMAP215 experiments, representative images are shown in Fig. 4c. Additional experiments were performed with 30 nM XMAP215 and lower γ-TuRC concentrations. All individual MTs in the field of view were counted in these conditions. 6 to 10-fold increase in nucleation was observed with XMAP215 and γ-TuRC combined, as compared to the sum of MTs generated by either component. Data from two (set 1) and three (set 2) independent γ-TuRC preparations was pooled. The number of fields of view analyzed (n): γ-TuRC (set 1)n = 20; 5 nM XMAP215 n = 20; γ-TuRC + 5 nM XMAP215 n = 20; γ-TuRC (set 2) n = 66; 30 nM XMAP215 n = 62; γ-TuRC + 30 nM XMAP215 n = 61. Mean ± s.d. of number of MTs was reported. (D) Fluorescence intensity measurements (displayed in Fig. 4d) is plotted for individual data points and color-coded by 3 experiments performed with independent γ-TuRC preparations. n = 10 randomly chosen fields of view assessed for every reaction and plotted individually. Means±s.d. of pooled data (n = 30 fields of view), overlaid in blue circles, is displayed in Fig. 4d. (E) MT nucleation was evaluated with γ-TuRC sample that was further purified using sucrose gradient fractionation after affinity step. Identical synergy with XMAP215 is seen with γ-TuRC purified using this scheme (compare to Fig. 4d). n = 10 fields of view were analyzed for each condition, except buffer (n = 9) and 130 nM XMAP215 (n = 11). Mean ± s.d. is plotted. The experiment was performed once supporting Fig. 4c-d. See Supplementary Fig. 9 for unprocessed scans, and Supplementary Table 2 for source data.