Anti β actin

Frozen tissues were homogenized in ice-cold lysis buffer. The protein concentration was determined by using Bicinchoninic Acid (BCA) Protein Assay Kit (Biorega, Tianjin, China) following the manufacturer's procedure. Equal amounts of protein (100 μg) were loaded and separated by SDS-PAGE gel, and then transferred onto polyvinylidene difluoride membrane. Membranes were blocked in 5% fat-free milk, and then incubated with anti-Foxp3 primary antibody (1:100; Biolegend) that was diluted in 5% fat-free milk. Anti–β-actin (1:500; Biolegend) was chosen as a standard. After three washes with PBS-T, membranes were incubated with appropriate horseradish peroxidase-conjugated antibody (ZSGB-BIO, Beijing, China), then washed again and developed with ECL Prime Western Blotting Detection Reagent (GE, Little Chalfont, Buckinghamshire, UK). The bands were scanned by Multispectral Imaging System (UVP, Upland, CA, USA) and analyzed by Quantity One software (Bio-Rad, Hercules, CA, USA).

Cells were lysed using a mammalian protein extraction reagent (M-PER; Thermo Scientific, Rockford, IL, USA) containing a protease inhibitor cocktail (Nacalai Tesque). Equal amounts of protein were resolved on 4–12% (w/v) gradient or 12% (w/v) SDS-PAGE gels and transferred to polyvinylidene fluoride membranes. The membranes were blocked and then incubated with the following primary antibodies: anti-LC3B (Cell Signaling Technology [CST], Danvers, MA, USA), anti-p62/SQSTM1 (CST), anti-Beclin-1 (CST), anti-Bcl-xL (BioLegend, San Diego, CA, USA), anti-caspase-3 (CST), anti-caspase-8 (Medical and Biological Laboratories, Nagoya, Japan), anti-caspase-9 (CST), anti-cyclin B1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-cFLIP_S/L (Santa Cruz Biotechnology), anti-XIAP (CST), anti-p21 (CST), anti-β-actin (BioLegend), and α-tubulin (Santa Cruz Biotechnology). After washing, the membranes were incubated at room temperature for 30 min with either goat anti-rabbit or goat anti-mouse alkaline phosphatase-conjugated secondary antibody (Invitrogen). Protein bands were visualized using the CDP-star chemiluminescence technique and imaged with the aid of an ImageQuant LAS-4000 system (FujiFilm, Tokyo, Japan). The band intensities were scanned and quantified using ImageJ software (http://rsb.info.nih.gov/ij/).

Lung protein was extracted as described above. Proteins were separated on stain-free gels (Bio-rad) and transferred onto polyvinylidene fluoride (PVDF) membranes and incubated with rabbit anti-STAT1 Ab (1:2000 dilution, Cell Signaling Technology) or with rabbit anti-phospho-STAT1 (Tyr701) Ab (1:2000 dilution, Cell Signaling Technology). Secondary Abs were goat anti-rabbit IgG (1:5000 dilution, Abcam). Anti-β-actin (1:2000 dilution, Biolegend) was incubated after stripping the STAT1 or P-STAT1 Ab. The relative amount of STAT1 and P-STAT1 protein was quantified by normalizing the amount of β-actin housekeeping protein using Image Lab software 6.1.

Cells were lysed using a mammalian protein extraction reagent (M-PER; PIERCE Scientific, Rockford, IL, USA) containing protease inhibitor cocktail (Nacalai Tesque). Equal amounts of protein were resolved on 4–12% gradient or 12% SDS-PAGE gels and then transferred to polyvinylidene fluoride membranes. After blocking the membranes, the blots were incubated with the following primary antibodies: anti-caspase-3 (9668: Cell Signaling Technology), anti-caspase-8 (M032-3; Medical and Biological Laboratories, Nagoya, Japan), anti-caspase-9 (9508; Cell Signaling Technology), anti-caspase-2 (2224; Cell Signaling Technology), anti-β-actin (BioLegend, San Diego, CA, USA) and anti-α-tubulin (Santa Cruz Biotechnology). Goat anti-rabbit or goat anti-mouse alkaline phosphatase-conjugated secondary antibodies (Invitrogen) were used to detect the primary antibodies. After washing, the membranes were incubated for 30 min at room temperature with an alkaline phosphatase-conjugated secondary antibody. Protein bands were visualized using CDP-star chemiluminescence and imaged using a LAS-4000 (FujiFilm, Tokyo, Japan).

Colons were flushed with PBS and frozen at –70 °C. Colon tissues were mechanically disrupted using the Bullet Blender® (Next Advance) at 4 °C and after lysis with RIPA buffer with Phosphatase Inhibitor Cocktail (PhosStop EASYpack, Roche) and Protease Inhibitor Cocktail (complete Tablets EASYpack, Roche). Tissues were centrifuged for 15 min at 4 °C and 16,000 g. Supernatants were collected, run on SDS gels, and transferred onto membranes. The membranes were blocked and probed with anti-Bcl-2 (Biolegend, San Diego, CA, USA) and anti-β-actin (Biolegend, San Diego, CA, USA). ImageJ was used for densitometry.

The 14-week-old Apc^Min/+ mice were immunized with VLP-hCEA orally. After 24 and 48 h, intestinal tumour polyps were collected and lysed for cellular protein extraction. Protein extractions were then subjected to SDS–polyacrylamide gel electrophoresis, electroblotted to a nitrocellulose membrane and immunoblotted against mouse caspase-1 p20 fragment (AdipoGen) or anti β-actin (Biolegend).