| Assay Method Information | |
| | Biochemical Assay |
| Description: | Compounds were tested employing a microtubule gliding assay, a standard biochemical assay for motor proteins. The first set of studies focused on human cytoplasmic dynein 2, the isoform involved in ciliary transport and Hedgehog signaling, as its inhibition in this assay has not been previously demonstrated. We purified an N-terminally GFP-tagged motor-domain construct of dynein 2 using an insect cell expression system [Schmidt et al., Nature. 2015; 518(7539):435-8]. The GFP tag in this construct allowed immobilization on passivated glass coverslips used in microtubule gliding assays. GFP-dynein 2, in the presence of ATP (1 mM), moved microtubules with a velocity of 128±14 nm/s. The mean velocity of dynein 2-driven microtubule gliding in the presence of control solvent (2% DMSO) and test compounds at 20 pM were compared. All motility assays were run at 1 mM MgATP, 0.05 mg/mL casein, and 2% DMSO. |
| Affinity data for this assay | |
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