Poly l lysine covered chamber slide

G-actin (1 mg/ml) Cytostebu-bio, Cytoskeleton) was incubated on ice for 1 hour and centrifuged at 4 °C for 20 min at 14000 g. HiLyteFluor 647-tubulin was purchased from Cytoskeleton and HyLight647-MT seeds were made as described in Mohan et al. 2013^{41 (link)} and stored at −80 °C. For the experiment seeds were quickly transferred into a 37 °C water bath, incubated for 20 min and kept in the dark at RT for 1–2 hrs. Labeled MTs were diluted 1:40 in PEM80 (80 mM PIPES, pH 6.9, 2 mM MgCl₂, 1 mM EGTA) containing 10 µM of taxol (Sigma). 8 µl Drebrin or DrebrinS142D (50 ng/µl), 1 µl EB3 (100 ng/µl), 1 µl ATP (5 mM), 1 µl GTP (20 mM), 2 µl taxol (200 µM), 2 µl 10x GT-buffer (800 mM PIPES pH 7.0, 20 mM MgCl₂, 5 mM EGTA), 2 µl Alexa-Fluor®488 labeled phalloidin (1:25 dilution), 2 µl G-actin (1 mg/ml; tebu-bio, Cytoskeleton), 1 µl MTs were added to a microtube, mixed, applied onto a poly-L-lysine covered chamber slide (Ibidi, Munich) and analyzed by TIRF-microscopy. TIRFM was performed on Visiscope Imaging system described above.

All reagents used for cell-free analysis of actin or MTs dynamics were from tebu-bio Cytoskeleton (Offenbach, Germany).
The effect of fascin on F-actin structure was analyzed by florescence microscopy: Actin (1 mg/ml) was incubated on ice for one hour and was then centrifuged for 15 min at 13.000 g, 4°C. The supernatant was used. 18 μl G-buffer (see [22 (link)]) including 200 μM ATP and 1 mM DTT, 2 μl actin, 2.5 μl Alexa-fluor^® conjugated phalloidin (1:25 dilution) as well as 2.5 μl 10 x assay buffer (see [22 (link)]) were mixed and applied to a poly-L-lysine-covered chamber slide (Ibidi, Martinsried, Germany). After about 20 min, actin bundles or cross links were monitored by fluorescence microscopy. Fluorescence intensity of actin bundles were analyzed by ImageJ. Five areas where actin bundles had been formed were analyzed per micrograph. In addition, five areas without actin bundles were analyzed (background). Then, mean values of fluorescence intensity of actin bundles and background were calculated and fluorescence intensity of actin bundles were subtracted from background. Five micrographs from two independent experiments were analyzed.
Actin bundling was measured by pull down experiments as described [22 (link)].