Table 6.

Summary of Dynein Adenosine Triphosphatase Activity Assay

Step	Parameter	Value	Description
1	Compound addition to plate	1 μL	0.5 or 2.5 mM compound in DMSO added to 384-well plates
2	Protein addition	42 μL	33.3 nM dynein in 20 mM Tris-HCl, pH 7.6, containing 50 mM KCl, 5 mM MgCl2
3	Incubation time	15 min	On ice
4	Warm samples	10 min	Lidded, 37°C
5	Preformed MT addition	2 μL	10 μM MTs stabilized by 10 μM PTX in assay buffer
6	ATP addition	5 μL	10 mM ATP in assay buffer
7	Incubation time	30 min	Lidded, 37°C
8	Malachite reagent addition	12.5 μL	PiColorLock™ Gold mixture
9	Incubation time	5 min	Lidded, ambient temperature
10	Stabilizer addition	5 μL
11	Assay Readout	630 nm	Absorbance

1. 0.5 or 2.5 mM compound used for 10 and 50 μM final concentrations, respectively.

5. For basal dynein assays, 2 μL buffer was used to replace MTs.

6. ATP prewarmed to 37°C before addition to plate.

10. Do not directly mix P_iColorLock™ Gold with stabilizer.

 PTX, paclitaxel.