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. 2021 Jan 8;134(1):jcs248815. doi: 10.1242/jcs.248815

Fig. 3.

Fig. 3.

UNC-45A breaks MTs in vitro independent of its C-terminal NMII-binding domain. (A) Schematic of full-length (WT) UNC-45A (1–944 aa), deltaN-UNC-45A (125–944 aa) or deltaC UNC-45A (1–544 aa). B. Recombinant full-length (WT) UNC-45A-GFP (130 kDa), deltaN UNC-45A-GFP (114 kDa) and deltaC UNC-45A-GFP (83.5 kDa) were separated on a 4–15% SDS gel, transferred onto PVDF membrane and blotted against a rabbit polyclonal antibody raised against full-length human UNC-45A. (C) Representative images of WT (top), C-terminally deleted (middle) or N-terminally deleted (bottom) UNC-45A (GFP-tagged) and rhodamine-labeled paclitaxel-stabilized MTs obtained via TIRF microscopy. (D) Tubulin (5 mg/ml) was incubated in the absence (vehicle) or presence of full-length (WT) UNC-45A (first panel), deltaN-UNC-45A (second panel) or deltaC-UNC-45A (third panel) or nocodazole (last panel) at the indicated concentrations at 4°C. MT polymerization was induced at 37°C and optical density was recorded over a period of 1 h. Results are expressed as absorbance as a percentage of control. All experiments were performed in triplicate.