JCS035006 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Neither acetylation of FAT10 nor catalytic activity of the CAT1 domain are required for its interaction with FAT10. (A) HEK293T cells were transfected with either catalytically active (wt) or inactive (mut) FLAG-CAT1 together with either wild-type or lysine-less (K0) HA-FAT10, followed by treatment with 5µM MG132 or DMSO for 6 hours. Cell lysates were subjected to anti-FLAG immunoprecipitation and subsequent analysis by western blot (WB). (B) GST-pulldown of an ³⁵S-methionine-labeled in vitro transcribed and translated active (wt) or inactive (mut) CAT1 domain by recombinant GST-FAT10.

• Supplemental Figure S2 -

  Fig. S2. Inhibition of catalytic activity has no effect on the interaction between HDAC6 and FAT10 in vitro. GST-pulldown of ³⁵S-methionine-labeled in vitro transcribed and translated HDAC6 with recombinant GST-FAT10 in the presence of 20 µM TSA or 160 µM tubacin.

• Supplemental Figure S3 -

  Fig. S3. FAT10 is acetylated but does not appear to be a substrate of HDAC6-mediated deacetylation. (A) HEK293T cells transfected with HA-FAT10 were treated with 5 µM MG132, 5 µM TSA or mock treated for 6 hours before lysis, immunoprecipitation (IP) with anti-FAT10 or control serum and subsequent analysis by western blot (WB) with an acetyl-lysine antibody (AcK). (B) HEK293T cells transfected with either HA-FAT10, FLAG-HDAC6 or empty vector were treated for 6 hours with 5 µM MG132 or DMSO before lysis, anti-FAT10 immunoprecipitation (IP) and analysis by western blot (WB). Asterisks denote the position of antibody light chains; arrows denote the position of HA-FAT10 on the anti-AcK western blots.