Mass Spectrometry Experiment 2 and 3: Comparison of Vpu WT and mutants.

HeLa P4.R5 cells were transfected to express Vpu constructs bearing C-terminal APEX2 tags, in duplicate. Following proximity biotinylation reactions, the biotinylated proteins were isolated and subject to quantitative mass spectrometry. Normalized TMT data are shown for duplicate experiments (Run 1 sample A and B, and Run 2 sample A and B, for each construct). The mean normalized TMT data were sorted into 6 k-means clusters (presented as a heatmap in Fig 6). The relative abundance of proteins was compared in Vpu WT vs. mutants pairwise, presented as log2 fold change Mutant/Vpu WT. Significance for proteomics data was assessed by Student’s t-test, p values of p ≤ 0.05 were considered significant. Volcano plots of pairwise analyses are presented in Figs 5 and S4. (XLSX)