Pore size nitrocellulose membrane

0.3 – 1 mg of soluble cell extract protein was incubated with 10 µl of GFP-Trap beads (ChromoTek, gta-10), M2 anti-FLAG Sepharose (Sigma, A2220), or PAWS1 antibody and Protein G-Sepharose (Sigma, P3296) for 1–2 h at 4°C with gentle agitation. Beads were then washed 5 times with lysis buffer. Immunoprecipitated proteins and protein extracts (10–20 µg) were denatured in SDS sample buffer and then separated by SDS-PAGE. Proteins were transferred to 0.2 µM pore size nitrocellulose membrane (GE Healthcare, 10600001). The membrane was blocked with 5% non-fat dry milk in TBS buffer (50 mM Tris–HCl, pH 7.5, 150 mM NaCl) or Odyssey Blocking Buffer in TBS (LI-COR, 927-50000) for 1 h and then with primary antibody diluted in blocking solution containing 0.1% (v/v) Tween-20 overnight at 4°C. Blots were incubated for 1 h at room temperature with the appropriate IRDye (LI-COR), StarBright (Bio-Rad) fluorescently conjugated or HRP conjugated secondary antibodies diluted in blocking solution, and visualised using the Odyssey Imager (LI-COR) or Chemidoc MP system (Bio-Rad, 17001402).

Western blot analyses of both cell lysates and EVs were carried out after resolving samples in 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In brief, the analysis on cell lysates was performed by washing cells twice with 1× PBS (pH 7.4) and lysing them with 1× SDS-PAGE sample buffer. Samples were resolved by SDS-PAGE and transferred by electroblotting on a 0.45 μM pore size nitrocellulose membrane (GE Healthcare Europe GmbH, Milan, Italy) overnight using a Bio-Rad (Hercules, CA, USA) trans-blot device. For Western blot analysis of EVs, they were lysed and analyzed as described for cell lysates. For immunoassays, membranes were blocked with 5% non-fat dry milk in PBS containing 0.1% Triton X-100 for 1 h at room temperature, then incubated overnight at 4 °C with specific antibodies diluted in PBS containing 0.1% Triton X-100. Filters were revealed using 1:1000-diluted sheep anti-Nef antiserum ARP 444 (MHRC, London, UK), 1:500-diluted anti-β-actin AC-74 mAb from Sigma (St. Louis, MI, USA), 1:500 diluted anti-Alix H-270 polyclonal Abs from Santa Cruz (Dallas, TX, USA), and 1:1000 diluted 6AT18 anti-6×His-tag mAb (Sigma).

Western blot analyses of both cell lysates and EVs were carried out after resolving samples in 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In brief, the analysis on cell lysates was performed by washing cells twice with 1× PBS (pH 7.4) and lysing them with 1 × SDS-PAGE sample buffer. Samples were resolved by SDS-PAGE and transferred by electroblotting on a 0.45 μM pore size nitrocellulose membrane (GE Healthcare Europe GmbH, Milan, Italy) overnight using a Bio-Rad (Hercules, CA, USA) Trans-Blot. For western blot analysis of EVs, they were lysed and analyzed as described for cell lysates. For immunoassays, membranes were blocked with 5% non-fat dry milk in PBS containing 0.1% Triton X-100 for 1 h at room temperature, then incubated overnight at 4 °C with specific antibodies diluted in PBS containing 0.1% Triton X-100. Filters were revealed using 1:1000-diluted sheep anti-Nef antiserum ARP 444 (MHRC, London, UK), 1:500-diluted anti-β-actin AC-74 mAb from Sigma (St. Louis, MI, USA), and 1:500 diluted anti-Alix H-270 polyclonal Abs from Santa Cruz (Dallas, TX, USA).