Western blotting was performed as previously described51 (link). Briefly, uninfected and HIV-1 infected or untreated and Meth treated CD4+ T-cells (after incubation period) were collected in cell lysis buffer, protein lysates were separated on NuPAGE precast gels (Life Technologies Corp.), transferred to 0.45 μm nitrocellulose membranes (Bio-Rad Laboratories, Hercules, CA), and probed with appropriate primary antibodies followed by incubation with their respective secondary antibodies. Proteins were visualized with Western Lightning Plus ECL Substrate (PerkinElmer, Waltham, MA).
For immunoprecipitation assay, CD4+ T-cells were left untreated or treated with Meth (100 μM) and incubated for times indicated. Cells were lysed with cell lysis buffer (Cell Signaling Technology). Cell lysates were subjected to immunoprecipitation using Millipore PureProteomeTM Protein A and Protein G Magnetic beads, which were used according to manufacturer’s protocol (MilliporeSigma, Burlington, MA) and the immune-complexes were further processed by Western blotting51 (link).
For immunoprecipitation assay, CD4+ T-cells were left untreated or treated with Meth (100 μM) and incubated for times indicated. Cells were lysed with cell lysis buffer (Cell Signaling Technology). Cell lysates were subjected to immunoprecipitation using Millipore PureProteomeTM Protein A and Protein G Magnetic beads, which were used according to manufacturer’s protocol (MilliporeSigma, Burlington, MA) and the immune-complexes were further processed by Western blotting51 (link).
Free full text: Click here
