Anti mouse igg hrp

The expression of COX-2, DR4 and DR5 proteins in monolayer and spheroids were detected by standard western blotting protocol. Briefly, cell lysates were separated by SDS-PAGE and transferred to a nitrocellulose blotting membrane (Bio-Rad, 162-0115). Membranes were blocked with 5% milk for 60 min at room temperature (RT, 25°C). The membranes were then incubated overnight at 4°C with 1∶2000 mouse anti-human COX-2 specific antibody (BD Biosciences, 610204), and 1∶2000 mouse anti-human DR4 and DR5 (BioLegend, 307201 and 307302), in 5% non-fat dry milk. Membranes were also probed with mouse anti-human β-actin (Santa Cruz biotechnology, sc-81178) at 1∶2000 dilution as a loading control. Following the overnight incubation with primary antibody, the membranes were washed with tris-buffered saline (TBS) with 0.1% Tween-20 and then incubated for 60 min at RT with 1∶1000 anti-mouse IgG-HRP (BD Biosciences, 554002) in 5% non-fat dry milk. The bands were visualized using a chemiluminiscent HRP substrate (Millipore, WBKL 50500) in a luminescent image analyzer (Fujifilm, LAS-4000). The level of PGE₂ in the cell culture media conditioned by cells propagating as tumor spheroids and monolayers was determined using Prostaglandin E₂ human ELISA kit (Invitrogen, KHL1701) following a protocol supplied by the manufacturer.

MG-63 and Saos-2 osteosarcoma cells were purchased from the American Type Culture Collection (Manassas, VA, USA) cultured in RPMI-1640 (Invitrogen Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 μg/ml streptomycin, in a 5% CO₂ humidified atmosphere at 37°C. α1-PDX (126850-2.5MG; Calbiochem-Merck KGaA, Darmstadt, Germany) was added to the medium at a concentration of 480 nM where indicated. Primary antibodies against Wnt, β-catenin, MT1-MMP and β-actin were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Other reagents were used, including anti-mouse-IgG-HRP and anti-rabbit-IgG-HRP (BD Biosciences, CA, USA) and Transwell invasion chambers (Promega, Madison, WI, USA).

Cells were seeded in six-well plates (2.5 × 10⁵/well for 3-h treatment; 2 × 10⁵/well for 24-h treatment), allowed to adhere for 24 h and treated with TRAIL or APG350 for either 3 or 24 h. Whole-cell lysates were prepared using RIPA buffer and analyzed by western blot as described^{23 (link)}. Primary and secondary antibodies used were purchased from: Cell Signaling Technology, Frankfurt/ Main, Germany (anti-caspase-8, anti-PARP, anti-phospho-p38, anti-phospho-IκBα, anti-phospho-p42/44, anti-phospho-JNK, anti-p38, anti-p42/44, anti-JNK, anti-mouse-IgG-HRP, anti-rabbit-IgG-HRP), BD Bioscience, Heidelberg, Germany (anti-Bcl-xL), R&D Systems, Minneapolis, Canada (anti-Bid), Santa Cruz, Heidelberg, Germany (anti-IκBa, anti-goat-IgG-HRP) and Sigma-Aldrich (anti-β-actin).