Figure 1.
Both Katanin subunits interact with microtubules. (A) Schematic of the in vitro crosslink assay. Briefly, purified proteins (KataninMEI-1-MEI-2 and microtubules) were mixed and incubated with EDC chemical crosslinker for 15 min. Samples were resolved on SDS PAGE and analyzed using stain-free (BioRad) based on tryptophan fluorescence labeling, Western blot, or mass spectrometry. (B) Crosslink assay between Katanin, composed of MEI-1 (orange) and MEI-2 full length (1–280) (purple) or N-terminal fragment (1–153) (light purple) and microtubules. Red stars indicate crosslinked complexes MEI-2*tubulin. SDS PAGE (BioRad) was analyzed by stain-free technique based on tryptophan fluorescence (Stain Free; Bio-Rad). (C) Crosslink assay between KataninMEI-1-MEI-2 or MEI-1 alone (orange) with or without GFP tag (green) and microtubules. Red stars indicate crosslinked complexes MEI-2*tubulin. Green star indicates the crosslinked complex GFP-MEI-1*tubulin. SDS PAGE was analyzed using tryptophan fluorescence (Stain Free; Bio-Rad) (upper panel) and by Western blot using anti-tubulin antibody (lower panel). Experiments were performed at least in triplicate and the maximal variation observed was <10%. Source data are available for this figure: SourceData F1.