Anti caspase 3 ab

Immunoblotting was performed as previously reported [13 ]. The transferred membranes were probed with the following primary polyclonal or monoclonal (m) antibodies (Abs): anti-p62/SQSTM1 (sc-28359), anti-GFP (sc-9996), and anti-β-actin (C4) mAbs (sc-47778) from Santa Cruz Biotechnology (Dallas, TX, USA); anti-poly ADP-ribose polymerase (PARP) Ab (#9542S) and anti-caspase-3 Ab (#9662) from Cell Signaling Technologies (Danvers, MA, USA); anti-LC3B Ab (NB600-1384) from Novus Biologicals (Littleton, CO); and anti-mCherry Ab (ab167453) from Abcam plc (Cambridge, UK). After incubation with the appropriate secondary peroxidase-conjugated Ab for 1–2 h, the immune-reactive proteins were detected using the Immobilon Western Chemiluminescent HRP Substrate (WBKLS0500, Millipore/Merck, Burlington, MA, USA). Densitometry was performed using a WSE-6300H/C LuminoGraph III (ATTO, Tokyo, Japan).

For TREM-2 expression, BMDMs were seeded in 6-well tissue culture plates and were infected with SeV at MOI 1 and 10 for 48 h. For caspase-3 expression, BMDMs were seeded in 6-well tissue culture plates and were cultured in the presence or absence of L cell-conditioned medium for 16 h. Cells were lysed and boiled in reducing SDS-PAGE sample buffer containing 100 mM Tris, pH 6.8, 4% SDS, 5% β-mercaptoethanol, 20% glycerol and 0.2% bromophenol blue. Lysates was subjected to 4–15% gradient gel electrophoresis (Bio-Rad Laboratories) and transferred to PVDF membrane (Millipore) in 10% methanol, 25 mM Tris and 192 mM glycine, pH 8.3. Anti-Caspase-3 Ab was from Cell Signaling Technology. Rat anti–mouse TREM-2 mAb (clone 178.9.4) was generated as described previously (Turnbull et al., 2006 (link)). Antibody binding was detected using the enhanced chemiluminescence (ECL) system (GE Healthcare). Molecular weight standards were run on each blot to validate that each of the indicated bands corresponded to the appropriate molecular weight.