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. 2018 Dec 3;217(12):4314–4330. doi: 10.1083/jcb.201712130

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© 2018 Wang et al.

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Figure 3. — K6a/K6b may regulate cell migration via interacting with myosin IIA. (A) Coimmunoprecipitation assay demonstrated that myosin IIA interacted with K6. n = 3 biological repeats. IB, immunoblot; IP, immunoprecipitation. (B) PLA showed that myosin IIA associated with K6. n = 4 biological repeats. Bar, 50 µm. The white values for all signals were set the same in WT and Krt6a/Krt6b-null keratinocytes. (C) Far-Western assay indicates that K6 may directly interact with myosin II via K6 head domain. Myosin II was the bait protein. Myosin IIA antibody was used to detect the binding of myosin II to proteins on the membrane. (D and E) Myosin IIA protein level was reduced in whole-cell lysates harvested from 6-d Krt6a/Krt6b-null skin explant culture. β-Actin was used as a loading control. Each bar represents the mean + SEM of six biological replicates. (F) The absence of K6a/K6b proteins did not affect the mRNA level of myosin IIA as measured by qRT-PCR with Gapdh and Rn18s as loading controls. Each bar represents the mean + SEM of three biological replicates. (G) Western blot analysis of whole-cell lysates harvested from 6-d skin explant culture indicated that adenovirus-delivered Cre (Ad-Cre)–induced Myh9 deletion in WT keratinocytes led to a decrease in DP protein level. Data represent the results of three biological repeats. (H) Cre-recombinase–mediated knockout of floxed-Myh9 did not result in increased connective cell migration (Video 6). Data represent the mean + SEM of four biological replicates. (I and J) PIV analysis indicated that neither speed nor angular deviation was affected by Cre-mediated knockout of Myh9. Experiments from the same day were paired and linked with a line. Paired difference was calculated by subtracting the value derived from GFP-expressing control cells from that of the Cre-expressing cells. (K) On type I collagen, MLCK inhibitor, ML-7, inhibited the migration of WT and Krt6a/Krt6b-null keratinocytes. Inhibition of myosin II by blebbistatin significantly decreased migratory potential of Krt6a/Krt6b-null keratinocytes but only minimally affected that of WT keratinocytes on type I collagen. Data represent the mean + SEM of four to five biological replicates. (L) On type IV collagen, blebbistatin reduced the migration of both WT and Krt6a/Krt6b-null keratinocytes. Data represent the mean + SEM of four biological replicates. *, P < 0.05; **, P < 0.01, Student’s two-tailed t test.