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. Author manuscript; available in PMC: 2010 Jul 2.
Published in final edited form as: Eur Biophys J. 2008 Nov 14;38(4):381–393. doi: 10.1007/s00249-008-0381-1

Figure 4.

Figure 4

A: SDS-PAGE of MT proteins

I: 3 × MTs (30 μg). II–IV: Separation of MT proteins by chromatography on phosphocellulose. II: pure tubulin (30 μg) eluted in the void volume. III: MAP2 + Tau (15 μg) eluted by 0.35M NaCl in buffer A. IV: MAP1 and MAP2 + proteins of low MW (20 μg) eluted by 1M NaCl in buffer A. V,VI: MAP2 and Tau separated by chromatography on ultrogel ACA 34 in buffer A. V: MAP2 (10 μg). VI: Tau (2.5 μg). Identical patterns in SDS-PAGE of heat-stable MAP2 and Tau were obtained (not shown).

B: MT gelation requires MAPs

I: Time-dependent viscosity changes at 35 °C of pure PC tubulin (6.4 mg/ml) in buffer B, incubated at 35 °C alone (open squares) or in the presence of either 0.14 mg/ml of the MAP fraction eluted from the PC column by 0.35M NaCl (MAP2 + Tau) (closed circles) or 15 μM taxol (dotted line, open stars). Values are the mean of duplicate assays.

II: Viscosity measured after 2 h at 35 °C of pure PC tubulin (6.4 mg/ml) incubated in the presence of increasing concentrations of the MAP fraction eluted from the PC column by 1M NaCl (MAP1 + MAP2). The value obtained for the same tubulin sample incubated for 2 h in the presence of 15 μM taxol is shown (open square). Values are the mean of duplicate assays.

C: Pure tubulin requires native MAP2 for the induction of a cAMP-dependent stimulated gelation

I: The viscosity of pure tubulin (4.9 mg/ml) purified according to (Castoldi and Popov, 2003), was measured after incubation for 30 min at 35 °C in buffer B, in the presence of increasing concentrations of native MAP2 (closed circles), thermostable MAP2 (dotted line, closed stars), native Tau (open circles), or thermostable Tau (dotted line, open stars). Values are the mean of duplicate assays.

II: Viscosity of pure tubulin (3.7 mg/ml) incubated for 30 min at 35 °C alone or in the presence of native MAP2 (0.15 mg/ml), thermostable MAP2 (0.15 mg/ml), or native or thermostable Tau (0.15 mg/ml). Samples contained 5 μM cAMP (hatched columns) or not (black columns). Assays were done in triplicate and values are shown +/− s.d.

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