Ecl detection kit

Huh-7 cells were seeded in 24-well plates at a density of 5 × 10⁴ cells per well overnight and treated with indicated reagent at proper concentrations for three days. Cells were washed with cold phosphate-buffered saline (PBS) and lysed by radioimmunoprecipitation assay (RIPA) lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 2 mM ethylenediaminetetraacetic acid (EDTA), 1 mM ethylene glycol bis(2-aminoethyl)tetraacetic acid (EGTA), 1 mM NaVO₃, 10 mM NaF, 1 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride (PMSF), 25 μg/mL aprotinin, and 25 μg/mL leupeptin) and stored at −20 °C. The protein concentration was determined by the Bradford method. Then, 10-μg protein samples were separated by 10% SDS-PAGE and transferred onto a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked with 5% non-fat dried milk and incubated with specific antibodies against GAPDH (1:10000, Genetex, Irvine, CA, USA) and COX-2 (1:1000; Cayman Chemical, Ann Arbor, ML, USA). Antibodies were diluted in 5% milk containing Tris-buffered saline (TBS) and 0.5% Tween. The blotting signal was developed using an enhanced chemiluminescence (ECL) detection kit (PerkinElmer, Waltham, MA, USA) and was counted by the software Quantity One (Bio-Rad, Hercules, CA, USA).

shRNA constructs were validated by western blotting. Nlgn- and shRNA-transfected HEK293T cells were solubilized in lysis buffer (10 mM Tris, pH 8.0, 200 mM NaCl, 1% Triton X-100, 1% SDS, and protease inhibitors) and loaded onto 8% SDS-PAGE gels. HEK293T cell line (Sigma) was authenticated by STR-PCR. Primary antibodies (1:1000 to 1:3000 dilution) were applied in blocking buffer (20 mM Tris, pH 7.4, 137 mM NaCl, 0.1% Tween 20, 1% bovine serum albumin, and 5% nonfat milk) for 2 hr at room temperature. Secondary antibodies were used at 1:2000 dilution. The signal was detected using an ECL detection kit (PerkinElmer Life Sciences).