AIPL1 alters the NUB1-mediated degradation of FAT10.
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SK-N-SH Cells were transfected with NUB1-FLAG, HA-FAT10 and Myc-AIPL1 vectors in the presence and absence of the proteasome inhibitor MG132, as indicated. Cell lysates were harvested 24 hours post-transfection and immunoprecipitates were analyzed by immunoblotting to detect the protein indicated. (A) NUB1 interacts with FAT10 and accelerates the degradation of free FAT10 and FAT10-modified proteins. The change in levels of FAT10 was measured from 3 independent experiments (n = 3) of duplicate samples. Heavy (h) and light (l) immunoglobulin chains are indicated. (B) NUB1 co-precipitates with AIPL1. (C) AIPL1 enhances the steady-state levels of free FAT10 and FAT10 modified proteins, both alone and in the presence of NUB1. The change in levels of FAT10 was measured from 5 independent experiments (n = 5) of duplicate samples. (D) HA-FAT10 was visualised by immunocytochemical analysis with anti-HA and Cy2-conjugated secondary antibody. NUB1-mediated degradation of FAT10 is altered by the presence of AIPL1. Scale bar is 20 µM. (C) and (E) A small proportion of AIPL1 is itself covalently modified with FAT10, as detected by anti-HA, anti-AIPL1 and anti-FAT10 (rabbit polyclonal) antibodies. The percentage of AIPL1 modified by FAT10 was measured from 3 independent experiments (n = 3). Conjugation of FAT10 to AIPL1 is prevented using a FAT10 diglycine deletion mutant. The position of molecular weight markers is indicated in kilodalton (kDa).