Nef induces CXCR4 ubiquitinylation mediated by the E3 ubiquitin ligase AIP4.

<p>A) Effect of proteosome inhibitors on Nef induced downregulation of CXCR4. Histograms showing relative (%) MFVs (with standard deviation) of native CXCR4 or CD4 (* p<0.01) in GFP gated Jurkat cells co-expressing GFP with Nef or null mutant and treated with the proteosome inhibitors lactacystin and epoxomicin at 25 µM (n = 4). <b>B</b>) Nef expression enhanced CXCR4 ubiquitinylation. HEK-293 cells were co-transfected with CD8, HA tagged CXCR4 and FLAG tagged ubiquitin with Nef or empty vector. Cytoplasmic extracts were prepared and an aliquot immuno-blotted to estimate HA-CXCR4 in the Nef (−) and Nef (+) transfectants (input lanes in B1). Ubiquitinylated proteins in the Nef (−) and Nef (+) cell lysates were affinity purified using FLAG mAb and immuno-blotted for CXCR4 using antisera against the HA epitope (<b>B1</b>). Molecular mass markers are denoted to the right of the gel in B1. Alternatively, ubiquitinylated proteins from Jurkat cells transfected Nef or null plasmid were captured using UBI-capture beads (Enzo Biochem) and resolved by SDS/PAGE followed by immuno-blot detection using anti-CXCR4 antibody (<b>B2</b>), Numbers to the left refer to molecular mass markers. The gel strip at the bottom illustrates immuno-blot analysis of aliquots (10%) of lysates (Input) for total CXCR4 content. (<b>C</b>) Nef recruits AIP4 to CXCR4 in the absence of receptor activation. Jurkat cells were co-tranfected with 6-His tagged CXCR4, GFP, FLAG-tagged wt or the Q297A/N329A AIP4 mutant that does not bind to CXCR4 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086998#pone.0086998-Bhandari1" target="_blank">[87]</a>, and Nef or a null plasmid. Cytoplasmic extracts were prepared after adjusting cell numbers to reflect equivalent GFP expression and aliquots were analyzed by SDS/PAGE and immuno-blot detection of AIP4 content using FLAG mAb (upper row). 6-His tagged CXCR4 proteins in the remaining extract were recovered by binding to Ni⁺⁺ NTA, resolved by SDS/PAGE, and immuno-blotted with FLAG mAb for detecting AIP4 (middle row). To assess transfected levels of AIP4, cell lysates were immuno-precipitated with rabbit polyclonal antibody against AIP4 followed by immuno-blotting with FLAG mAb (lower row). Nef functionality was evaluated by monitoring CD4 expression on transfected cells by flow cytometry (histogram below, * p<0.01).