Figure 4: p68 and CaM interact with microtubules.

(a,b) Precipitation of HA-p68 that was immunopurified from cell lysates of multiple scratch-wound-treated (scratch) or untreated (non scratch) SW480 cells with in vitro assembled microtubule (pellet) in the presence of CaM (p68/CaM) was probed by immunoblot of the precipitated pellets using the anti-HA antibody (IB:HA). The precipitations were carried out in the presence of 0.5 mM CaCl₂ (Ca²⁺) or 5 mM EGTA (EGTA) (a), or in the presence (ATP) and absence (−ATP) of ATP or in the presence of non-hydrolysable ATP analogue AMP-PNP (AMP-PNP) in addition to the presence of 0.5 mM CaCl₂ (b). Immunoblot analyses of α-tubulin (IB:Tubulin) is the loading control indicating amounts of precipitated microtubule. (c) Representative fluorescence microscopy images reveal the bindings of rhodamine-labelled microtubule (rhodamine MT) to the glass slides on which the HA-p68 that was immunopurified from cell lysates of multiple scratch-wound-treated (scratch) or untreated (non scratch) SW480 cells was attached (in the presence of CaM and Ca²⁺ [p68/CaM] and absence of CaM [p68]). (d) Representative fluorescence microscopy images of SW480 cells that expressed DsRed-p68 and eGFP-α-tubulin. The cells were treated by scratch-wound. The arrows indicated the DsRed-p68. (e) ATPase activities of HA-p68 or mutant (indicated) that was immunopurified from cell lysates of multiple scratch-wound-treated SW480 cells in the presence of various different substrates (indicated) were analysed by measuring inorganic phosphate released from ATP hydrolysis. The ATPase activities were measured in the presence of 0.5 mM CaCl₂ (Ca²⁺) or 5 mM EGTA (EGTA), and in the presence and absence of the recombinant CaM. In the ATPase assays, 4 mM ATP was always added unless specified (-ATP). The ATPase activity was expressed as μmole of inorganic phosphate hydrolyzed from ATP. MT is in vitro assembled microtubule. RNA is total RNA extracts from yeast. Scale bars, 5 μm (for c) and 10 μm (for d).