Fig. 1. HDAC6 interacts with FAT10 only under proteasome inhibition and has no influence on the degradation rate of FAT10. (A) HEK293T cells, transiently transfected with HA-FAT10 and either wild-type (wt) FLAG-HDAC6, a BUZ mutant or empty vector (–), were treated with 5 µM of the proteasome inhibitor 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 were transiently transfected with FLAG-HDAC6 followed by treatment with 500 U/ml TNF- and 200 U/ml IFN- for 16 hours, and 5 µM MG132 for another 6 hours before lysis, anti-FLAG immunoprecipitation (IP) and analysis by western blot (WB). (C) HEK293T cells transfected with either HA-FAT10 alone (left panel), HA-FAT10 and FLAG-HDAC6 together (center panel) or FLAG-HDAC6 alone (right panel) were pulse-labeled with [³⁵S]-methionine and chased for the indicated times (hours). Cell lysates were subjected to anti-HA and anti-FLAG immunoprecipitation followed by SDS-PAGE and autoradiography.