Supplementary Figure 8: XMAP215 interacts with γ-tubulin and αβ-tubulin with distinct termini, Related to Figs. 7 and 8a.

(A) Size exclusion chromatography of 140 nM human γ-tubulin was performed in buffer containing 400 mM KCl to prevent γ-tubulin oligomerization. In high salt, γ-tubulin elutes in fractions L-M (marked [**], compare to first run in Fig. 7). This experiment was performed once at the specified condition, with three supporting runs at slightly different concentration and buffer conditions. (B) Immunoprecipitation of γ-tubulin by XMAP215 in vitro. GFP or XMAP215-GFP at 500 nM each, or buffer were mixed with 80 nM human γ-tubulin and precipitated using anti-GFP antibodies. γ-tubulin content co-precipitated was analyzed via immunoblot. The experiment was repeated independently three times. (C) Size exclusion chromatography was performed with 140 nM γ-tubulin and 1.2 μM TOG1212 or 0.7 μM XMAP215 FL 1-5AA. 500 μl sample volume was injected into the column, and 300 μl fractions were collected. Alternate fractions eluted between 8.5 ml to 16.6 ml were analyzed via SDS-PAGE followed by immunoblot with γ-tubulin antibodies and GFP antibodies to detect the elution profile of γ-tubulin and GFP labeled XMAP215 proteins, respectively. Shift in γ-tubulin signal was observed. Each chromatography run was repeated at least twice on different days at the specified concentration, and two supporting runs for TOG1212 at slightly different protein concentrations were performed. (D) Wild-type XMAP215 (4.3 μM) and bovine αβ-tubulin (4.4 μM) were mixed and applied to size exclusion chromatography. 100 μl sample volume was injected into the column, 200 μl fractions were collected and alternate fractions eluted between 8.5 ml to 13.9 ml were analyzed via SDS-PAGE. Elution fractions for full-length XMAP215, N-terminal TOG1-4 construct or C-terminal TOG5-CT construct with or without bovine αβ-tubulin were analyzed. Stoke’s radii of reference proteins are marked at their peak elution: Thyroglobulin (8.6 nm) and Aldolase (4.6 nm). Each chromatography run was performed once at the specified conditions. Two supporting runs for full-length XMAP215 and one for TOG5-CT performed under slightly different concentrations and buffers. Void volume of the column was measured as 8.7-8.8 ml. See Figs. 7 and 8a and Supplementary Fig. 9.