Hrp conjugated goat anti mouse igg

Mouse antibody against ASAH1 (612302) was purchased from BD Biosciences (San Jose, CA). Anti-actin, carmofur, temozolomide (TMZ), and N-oleoylethanolamine (OE), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma Aldrich (St. Louis, MO). DMSO, used to dissolve carmofur, temozolomide, and OE, was purchased from Sigma Aldrich (St. Louis, MO). HRP-conjugated goat anti-mouse IgG was supplied by R&D Systems, Inc. (Minneapolis, MN). SDS-PAGE and Western blotting materials and the Annexin V kit were obtained from Life Technologies, Inc. (Grand Island, NY).

Cells were collected and lysed in hypotonic buffer with nonionic detergent (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5% NP-40, 50 mM NaF with 0.5 mM phenylmethylsulfonyl fluoride), incubated on ice for 15 min and cleared by centrifugation at 10,000 x g at 4°C for 10 min. Protein concentration was determined using a Bradford assay (Bio-Rad Laboratories, Hercules, CA, USA). Equal amounts of protein were mixed with reducing Laemmli loading buffer, boiled and electrophoresed on NuPAGE 4-12% Bis-Tris Gels (Invitrogen, Carlsbad, CA), then transferred electrophoretically to nitrocellulose membranes in Tris-glycine buffer. The membranes were blocked with 5% nonfat dry milk in TBST (with 0.05% Tween 20) for 1 h at RT, and then incubated with 1 μg/ml primary antibodies at 4°C overnight. After washing five times (5 min each wash) with TBST buffer, the membranes were incubated with HRP-conjugated goat anti-mouse IgG (1:10,000 dilution, R&D Systems, Minneapolis, MN) for 1 h at RT. The reactions were detected by using an ECL Western blot detection kit (Thermofisher, Waltham, MA) and the images were recorded by VersaDOC imaging system (BioRad, Hercules, CA).