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. 2014 Jan 6;204(1):19–28. doi: 10.1083/jcb.201306083

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© 2014 Chan et al.

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Figure 3. — ADD1 phosphorylation at S12 and S355 is required for its interaction with the motor domain of Myo10. (A) The cell lysates prepared from HeLa cells or those stably expressing FLAG-ADD1 were incubated with anti-FLAG M2 affinity resins. The bound proteins were eluted from the resins with FLAG peptides and analyzed by immunoblotting (IB) with anti-Myo10 or anti-FLAG. WCL, whole-cell lysates. (B) GFP-fused Myo10 (GFP-Myo10) and mutants were transiently expressed in HEK293 cells and were analyzed by immunoblotting with anti-GFP. The scheme shows the domain structures of GFP-Myo10. CC, coiled-coil domain; PEST, polypeptide enriched in proline, glutamic acid, serine, and threonine residues. (C and D) FLAG-ADD1 was transiently coexpressed with GFP-Myo10 or its mutants in HEK293 cells. GFP-Myo10 was immunoprecipitated (IP) by anti-GFP, and the immunocomplexes were analyzed by immunoblotting with anti-FLAG or anti-GFP. (E) GFP-Myo10-N was transiently coexpressed with (+) or without (−) the FLAG-ADD1Δtail mutant in HEK293 cells. The cell lysates were incubated with anti-FLAG, and the immunocomplexes were analyzed by immunoblotting with anti-GFP or anti-FLAG. (F) GFP-Myo10-N was transiently coexpressed with FLAG-ADD1 or its mutants in HEK293 cells. FLAG-ADD1 was immunoprecipitated by anti-FLAG, and the immunocomplexes were analyzed by immunoblotting with anti-GFP or anti-FLAG. (G) HeLa cells were infected with lentiviruses expressing shRNAs to Myo10 (sh-Myo10 #1 and sh-Myo10 #2) or to luciferase (sh-Luc.) as a control. The expression levels of Myo10, ADD1, and β-tubulin (as a loading control) were analyzed by immunoblotting with the indicated antibodies. (H) The cells, as in G, were stained for ADD1, α-tubulin, and DNA. Arrows indicate misaligned chromosomes. The percentage of ADD1 association with mitotic spindles in the total number of mitotic cells counted was measured (n > 270). Values (means ± SD) are from three independent experiments. **, P < 0.01. Bars, 5 µm.