10.1371/journal.pone.0030866.g001 John S. Bett John S. Bett Naheed Kanuga Naheed Kanuga Emma Richet Emma Richet Gunter Schmidtke Gunter Schmidtke Marcus Groettrup Marcus Groettrup Michael E. Cheetham Michael E. Cheetham Jacqueline van der Spuy Jacqueline van der Spuy AIPL1 alters the NUB1-mediated degradation of FAT10. Public Library of Science 2013 alters nub1-mediated degradation 2013-02-20 09:16:31 Figure https://plos.figshare.com/articles/figure/_AIPL1_alters_the_NUB1_mediated_degradation_of_FAT10_/355733 <p>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).</p>