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. 2017 Jan 4;292(8):3099–3111. doi: 10.1074/jbc.M116.733402

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© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

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FIGURE 4. — NMIIA phosphorylation is not critical for 2D migration in 4T1 mammary gland carcinoma cells. A, whole-cell lysates of parental 4T1 cells, MHC-IIA shRNA cells, and MHC-IIA shRNA cells expressing the indicated GFP MHC-IIA construct were subjected to Western blotting analysis with anti-MHC-IIA and anti-GFP antibodies. B, 4T1 cell series was analyzed for 2D migration using a modified scratch wound assay on collagen I-coated glass surfaces and time-lapse imaging. Sample images from parental GFP MHC-IIA-expressing and GFP MHC-IIA 3×A-expressing 4T1 cells are shown at t = 0 (left column) and t = 12 h (right column). C, quantification of 2D migration derived from time-lapse imaging reveals modest change upon MHC-IIA shRNA or upon re-expression of NMIIA phosphorylation site mutants. Data were plotted as mean ± S.D., n = 9 fields of view, with three fields of view quantified from each of three separate experiments performed on different dates. Migration time points were analyzed by one-way analysis of variance (Tukey-Kramer test).