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. 2016 May 26;7(25):37471–37486. doi: 10.18632/oncotarget.9650

Figure 4. HDAC4 mediates Osx deacetylation.

Figure 4

A. HEK 293T cells were transiently transfected with the HA-Osx-GFP expression plasmid alone or together with Flag-HDAC1, Flag-HDAC3, Flag-HDAC4, or Flag-HDAC5 expression vectors. The Osx protein was detected by western blotting with an anti-HA antibody. β-Actin served as a loading control. B. HEK 293T cells were transiently co-transfected with Flag-CBP-HA, HA-Osx-GFP and Flag-HDAC4 or not, the cell lysates were immunoprecipitated with an anti-HA antibody and then blotted with an anti-Acetylated-Lys antibody. Flag-CBP-HA and Flag-HDAC4 protein were detected by western blotting with an anti-Flag antibody. HA-Osx-GFP protein was detected with an anti-HA antibody. C. HEK 293T cells were transiently transfected with HA-Osx-GFP alone or together with Flag-HDAC4 expression plasmid, the cell lysates were immunoprecipitated with an anti-Flag antibody and then blotted with an anti-HA antibody. Flag-HDAC4 and HA-Osx-GFP protein were detected by western blotting with an anti-Flag and anti-HA antibody, respectively. D. HEK 293T cells were transiently transfected with Flag-HDAC4 alone or together with the HA-Osx expression plasmid, the cell lysates were immunoprecipitated with an anti-HA antibody and then blotted with an anti-Flag antibody. Flag-HDAC4 and HA-Osx protein were detected by western blotting with an anti-Flag and anti-HA antibody, respectively. E. HEK 293T cells were transiently co-transfected with HA-Osx-GFP and Flag-HDAC4 expression plasmids. The localization of Osx was visualized as green fluorescence, the localization of HDAC4 was visualized by immunostaining with an anti-Flag antibody (red). Nuclei were visualized by DAPI staining (blue). Experiments were repeated at least three times.