Anti actin ac 74 mab

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 (Amersham) overnight using a Bio-Rad Trans-Blot. EVs 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 1:1,000diluted sheep anti-Nef antiserum ARP 444 (MHRC, London, UK), 1:500-diluted anti--actin AC-74 mAb from Sigma, 1:500 diluted anti-Alix H-270 polyclonal Abs from Santa Cruz, and 1:1,000 diluted anti-flag M2 mAb from 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 1x SDS-PAGE sample buffer. Samples were resolved by SDS-PAGE and transferred by electroblotting on a 0.45 μM pore size nitrocellulose membrane (Amersham) overnight using a Bio-Rad 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 1:1,000-diluted sheep anti-Nef antiserum ARP 444 (MHRC, London, UK), 1:500diluted anti--actin AC-74 mAb from Sigma, and 1:500 diluted anti-Alix H-270 polyclonal Abs from Santa Cruz.

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 1x SDS-PAGE sample buffer. Samples were resolved by SDS-PAGE and transferred by electroblotting on a 0.45 μM pore size nitrocellulose membrane (Amersham) overnight using a Bio-Rad 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 1:1,000-diluted sheep anti-Nef antiserum ARP 444 (MHRC, London, UK), 1:500diluted anti--actin AC-74 mAb from Sigma, and 1:500 diluted anti-Alix H-270 polyclonal Abs from Santa Cruz.