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. Author manuscript; available in PMC: 2015 Aug 1.
Published in final edited form as: Parkinsonism Relat Disord. 2014 May 13;20(8):884–888. doi: 10.1016/j.parkreldis.2014.05.004

Figure 2. Microtubule Binding assay for p.Y78C, p.G71R, p.G59S, p.Q74P DCTN1 mutants.

Figure 2

HEK293E cells were transfected with either wild-type or mutant DCTN1-V5, as indicated. To test the affinity to microtubules, equal amounts of lysates were incubated with assembled microtubules in vitro, placed on a glycerol cushion and separated via centrifugation at 100,000g. Supernatant and pellet were subjected to SDS-PAGE, blotted and analyzed with antibodies against V5 and β-tubulin. (a) Shown is a representative Western Blot of supernatant (middle, top) and pellet (middle, bottom). Input samples are indicated to the left. Equal loading is demonstrated by GAPDH probing. (b) Shown is the densitometric ratio of supernatant without microtubules to supernatant with microtubules as the mean value of five independent experiments. In contrast to wild-type DCTN1-V5, mutant DCTN1-V5 show considerable less microtubule binding, being significant for p.G71R, p.Q74 and p.Y78C. *: p<0.05, ns: not significant.