Supplementary Figure 3: γ-TuRC is necessary for microtubule nucleation with XMAP215, Related to Fig. 3.

(A) Western blot analysis of γ-tubulin and GCP4 (core γ-TuRC subunit) in γ-tubulin depletion. XMAP215, augmin (HAUS6), TPX2 and TACC3 were verified to be present in γ-tubulin depleted extracts. The experiment was repeated with three independent extract preparations, including verification with a different γ-tubulin antibody (XenC) for γ-TuRC immunodepletion. See Supplementary Fig. 9 for unprocessed scans. (B-C) Total number of MTs and fraction of MTs generated via branching MT nucleation in control and γ-tubulin depleted extracts was plotted with time for increasing concentration of excess XMAP215. Few MTs that emerged in γ-tubulin depleted extracts were counted manually (MTs generated by both de novo nucleation and branching nucleation). For IgG depletion, EB1 comets were counted as a readout for total number of MTs in the field of view (see Methods), while MTs nucleated de novo were counted manually. The number of branched MTs was calculated by subtracting MTs nucleated de novo from total number of MTs. The fraction of branched MTs for IgG depletion was then calculated as the ratio of MTs generated by branching to total EB1 comets. See Fig. 3, and Supplementary Video 5. The analyses were repeated twice on experiments performed with independent extract preparations. (D) De novo nucleation events observed by adding 360 nM XMAP215-GFP in excess of the endogenous protein to control and γ-tubulin depleted extracts without RanQ69L. Representative images are displayed at 16 minutes of the reaction. Scale bar, 10 μm. The experiment was repeated three times with independent extract preparations. (E) EB1 comets were detected and tracked using the image analysis procedure described in Methods. The number of MT tracks was plotted over time. MT-independent nucleation was severely reduced in γ-tubulin depletion, in comparison to the control depletion, even after the addition of excess XMAP215. The analysis was repeated thrice on experiments performed with independent extract preparations.