Chemilumi one chemiluminescent kit

Cells infected with AdV and AAV expressing HA-tagged iCaspase9 were lysed in Cell Lysis Buffer M (WaKo, Osaka, Japan). iCaspase9 in the lysates was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins in the gel were electrophoretically transferred to a membrane (Immunobilion; Millipore, Billerica, MA, USA). Blots were blocked and probed with anti-HA high affinity rat monoclonal antibody (Roche, Mannheim, Germany) and anti-actin rabbit polyclonal antibody (Abcam, Cambridge, UK) overnight at 4 °C. Blots were then incubated with peroxidase-conjugated anti-rat IgG (Jackson Lab, Bar Harbor, ME, USA) and anti-rabbit IgG (Jackson Lab), and bound antibodies were visualized using a Chemilumi-One Chemiluminescent Kit (Nacalai Tesque, Kyoto, Japan).

Cells were lysed in lysis buffer (50 mM Tris-HCl, 1% SDS, 2 μg/mL leupeptin, 2 μg/mL aprotinin, 0.5 mM PMSF, and 1 mM DTT). The lysate was sonicated and centrifuged at 15,000 g for 20 min at 4°C, and the supernatant was collected. The protein extract was loaded onto a 7.5, 10, or 12.5% SDS-polyacrylamide gel for electrophoresis, and blotted onto polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). Rabbit polyclonal anti-DR4, anti-DR5 (Prosci, Poway, CA, USA), and anti-CDK4 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and rabbit monoclonal anti-Bim (Epitomics, San Diego, CA, USA), and mouse monoclonal anti- cyclin D1 (MBL, Nagoya, Japan) and anti-β-actin (Sigma) antibodies were used as the primary antibodies. The blots were incubated with the appropriate HRP-conjugated secondary antibody (GE Healthcare, Piscataway, NJ, USA), and signals were detected using a Chemilumi-one chemiluminescent kit (Nacalai Tesque, Kyoto, Japan).

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.