Mouse anti human β actin

The treated cells were lysated with RIPA buffer (Abcam, Cambridge, UK) and collected for western blot. Protein quantification was performed using BCA kit (Abcam, Cambridge, UK). Protein (10 μg) was loaded for electrophoresis and transferred to PVDF membranes. The blots were blocked and labeled with primary antibodies (mouse anti-human HAT1, 1:2000 dilution; Santa Cruz Biotechnology, Dallas, TX, USA); rabbit anti-human KAT8, 1:2000 (Abcam, Cambridge, UK); or mouse anti-human β-actin 1:5000 (Santa Cruz Biotechnology, Dallas, TX, USA) overnight. Membranes were incubated with the secondary antibody (goat anti-rabbit-HRP or goat anti-mouse-HRP, 1:5000 dilution each; Amersham Pharmacia Biotech, Saclay, France). The signal was developed and imaged using ImageQuant™ LAS 4000 (Fujifilm, Tokyo, Japan).

The cells (1×10⁶) were washed with cold PBS and lysed in 100 μl lysis buffer [150 mmol/l NaCl, 20 mmol/l Tris-HCl (pH 7.5), 1 mmol/l EDTA, 1 mmol/l EGTA, 1 mmol/l Na₃VO₄, 1 mmol/l sodium fluoride, 0.5% DOC, 1% Triton X-100 and 1% Nonidet-P40]. The cell lysates were boiled with 2X loading buffer for 20 min and analyzed by western blotting. Mouse anti-human Runx3, mouse anti-human β-actin and anti-NF-κB were used as primary antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). Following polyacrylamide gel electrophoresis, the protein was transferred onto a PVDF membrane (Perkin-Elmer, Waltham, MA, USA). The membrane was incubated with the primary antibody, followed by incubation with horseradish peroxidase-conjugated rabbit anti-mouse IgG (Takara Bio, Inc., Shiga, Japan). After being thoroughly washed in Tris-Buffered Saline with Tween-20, the blots were processed for detection of Ag using an electrochemiluminescence plus western blotting detection system (GE Healthcare Life Sciences, Pittsburgh, PA, USA). The Typhoon Molecular Imaging system (GE Healthcare Life Sciences) was used for scanning and recording the results.

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.

The cytoplasmic and membrane protein fractions from treated human peritoneal macrophages and homogenized mouse liver tissues were isolated using extraction reagents according to the manufacturer's protocol (KeyGEN Biotech, Nanjing, China). The protein concentrations in all of the samples were determined by using the Bradford Protein Assay Kit (KeyGEN Biotech, Nanjing, China). Western blotting was performed as described previously [9 (link)]. The following antibodies were purchased from Santa Cruz (CA, USA): mouse anti-human TLR2, mouse anti-human TLR4, mouse anti-human MR, mouse anti-human MyD88, rabbit anti-mouse MyD88, rabbit anti-mouse MR, rabbit anti-mouse β-actin, and mouse anti-human β-actin. Immunoreactivity was detected using a horseradish peroxidase-conjugated second antibody and an enhanced chemiluminescence reaction (Pierce, Rockford, IL, USA).