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Fig. 4. Neither acetylation of FAT10 nor catalytic activity of HDAC6 are required for interaction between the two proteins. (A) HEK293T cells transfected with HA-FAT10 and either wild-type (wt) FLAG-HDAC6 or a catalytically dead mutant (mut) were treated with 5 µM MG132, or DMSO as a negative control, for 6 hours before lysis, anti-FLAG immunoprecipitation (IP) and analysis by western blot (WB). (B) HEK293T cells transfected with HA-FAT10 and FLAG-HDAC6 were treated with 5 µM MG132, 1 µM nocodazole, 5 µM of the HDAC inhibitor trichostatin A (TSA) or mock treated for 6 hours before lysis, anti-FLAG immunoprecipitation (IP) and analysis by western blot (WB). (C) HEK293T cells transfected with HA-FAT10 and FLAG-HDAC6 were treated with 5 µM MG132, 20 µM tubacin or mock treated for 6 hours before lysis, anti-FLAG immunoprecipitation (IP) and analysis by western blot (WB). (D) HEK293T cells, transfected with FLAG-HDAC6 and either wild-type (wt) or a lysine-less HA-FAT10 mutant (K0), were treated with 5 µM MG132 or DMSO for 6 hours before lysis, anti-FLAG immunoprecipitation (IP) and analysis by western blot (WB). (E) HEK293T cells transfected with HA-FAT10 and FLAG-HDAC6 were treated with 5 µM MG132, 5 µM TSA or mock treated for 6 hours before lysis, anti-FLAG immunoprecipitation (IP) and analysis by western blot (WB) with an acetyl-lysine antibody (AcK). The asterisk denotes the position of the antibody light chain; the arrowhead denotes the position of HA-FAT10 on the anti-AcK western blot.
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