Figure 3.

Detection of Nef^mut/SARS-CoV-2 fusion products in transfected cells and extracellular vesicles (EVs). Western blot analysis of 30 μg of lysates from HEK293T cells transfected with DNA vectors expressing Nef^mut fused with either S1, S2, M, or N SARS-CoV-2 open reading frames (ORFs) (left panels). Equal volumes of buffer where purified EVs were resuspended after differential centrifugations of respective supernatants were also analyzed (right panels). As control, conditions from mock-transfected cells as well as cells transfected with a vector expressing Nef^mut alone were included. Polyclonal anti-Nef Abs served to detect Nef^mut-based products, while β-actin and Alix were revealed as markers for cell lysates and EVs, respectively. Relevant signals are highlighted. Molecular markers are given in kDa. Relative intensities of relevant signals after normalization with either β-actin (for cell lysates) or Alix (for EVs) signals were also reported as arbitrary units (A.U.), and considering the Nef^mut conditions as reference samples. The results are representative of four independent experiments (Figure S1).