Peroxidase linked protein a

Monocytes and macrophages (3.5×10⁶ cells) were lysed in Laemmli sample buffer (100 mM Tris-HCl, pH 6.8, 0.04% sodium dodecyl sulfate (SDS), 20% glycerol, 0.12% 2-mercaptoethanol). Proteins in the lysates were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins in the gel were then electrically transferred to a membrane (Immobilion; Millipore, Billerica, MA). Blots were blocked and probed with anti-CypA affinity rabbit polyclonal antibody (Sigma, St. Louis, MO) overnight at 4°C. Blots then were incubated with peroxidase-linked protein A (GE Healthcare, Buckinghamshire, UK), and bound antibodies were visualized with a Chemilumi-One chemiluminescent kit (Nacalai Tesque, Kyoto, Japan). Quantities of cell lysate were normalized by CypA level, then subjected to a new round of SDS-PAGE and membrane transfer. For the new blot, SAMHD1 protein in the membrane was detected with anti-SAMHD1 (611–625) rabbit antibody (Sigma) followed by peroxidase-linked protein A (GE Healthcare, Buckinghamshire, UK) detection as described above.