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. 2013 Dec 1;24(23):3651–3662. doi: 10.1091/mbc.E13-05-0277

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© 2013 Seldin et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 6: — Anaphase localization of NuMA is independent of LGN and 4.1 binding. (A–D) Immunofluorescence analysis of endogenous NuMA localization in wild-type and LGN-knockdown keratinocytes in metaphase and anaphase, as indicated. (E) Quantitation of cells with cortical NuMA accumulation, as indicated. n = 50 cells, p < 0.0001 for NuMA localization in WT vs. LGN KD cells in metaphase. (F–I) Epifluorescence of NuMA-GFP and NuMAΔLGN–BD-GFP transfected keratinocytes in metaphase and anaphase, as indicated. (J) Quantitation of cells with cortical NuMA accumulation, as indicated. n = 25 cells, p < 0.0001 for NuMA-GFP vs. NuMAΔLGN BD-GFP cortical localization in metaphase. (K, L) Immunofluorescence analysis of endogenous NuMA localization in LGN-knockdown keratinocytes treated with DMSO or 100 μM purvalanol A for 3 min. (M) NuMA construct that harbors the T2055A mutation and spans from the carboxy-terminal 4.1-binding domain to the end of the protein. (N) A GFP-tagged version of the NuMA fragment diagrammed in M was expressed in wild-type cells, and immunofluorescence analysis was performed to determine localization. (O) Quantitation of cells from K and L with cortical NuMA accumulation, as indicated. n = 50 cells, p < 0.0001. (P–S) GFP epifluorescence of NuMA constructs as indicated. The domain structure of each NuMA fragment is diagrammed beneath its corresponding image. (T) Quantitation of the ability of NuMA deletion/mutant constructs from N and Q–S to target to the cell cortex in metaphase. n = 25 cells, all p > 0.05. (U) Immunoprecipitation of GFP-tagged NuMA constructs/mutants with 4.1G-HA. Top and bottom, amount of 4.1G-HA and GFP fusion proteins in the immunoprecipitates, respectively.