Anti lc3

For Western blotting, cell lysates in ice-cold RIPA buffer were centrifuged and the supernatants were assayed for protein content. About 20 to 30 μg of proteins were fractionated on 7.5–15% polyacrylamide gel and transferred onto PVDF membrane from Millipore Corporation (Billerica, MA, USA). Nonspecific binding sites were blocked for 1 hour at RT in 20 mM Tris-HCl (pH 7.4) buffer, 55 mM NaCl and 0.1% Tween 20 containing 5% non-fat dry milk (blocking buffer). Membranes were then incubated overnight at 4°C with primary antibody diluted in blocking buffer. Primary antibodies used are anti-LC3, anti-cyclin D1, anti-p62, anti-actin and anti-pERK1,2 antibodies from Sigma Aldrich (St. Louis, USA), anti-p53 and anti-pEGFR antibodies from Santacruz Biotectnology (SantaCruz, CA, USA), anti-cyclin B2 was from Abcam (Cambridge Science Park, Cambridge, UK). All these antibodies are dissolved in blocking solution. After extensive washings and incubation with the respective horseradish peroxidase-labeled secondary antibodies, protein presence was visualized by enhanced chemiluminescence reaction from Pierce Biotechnology (Rockford, IL, USA). Band relative densities obtained using Alliance 4.7 UVITEC (Cambridge, UK) were normalized to actin and values were given as relative units (R.U.).

Extraction of proteins from mouse colonic mucosa and Western blot analysis were performed as previously described [40 (link),41 (link)]. The primary antibodies used were anti-LC3 (#L8918, Sigma-Aldrich, Saint-Louis, MO, USA), anti-phospho-H2AX (#2577, Cell Signaling) and anti-α-tubulin (#2144, Cell Signaling). The secondary antibody used was HRP-conjugated anti-rabbit (#7074, Cell Signaling). Blots were detected using the Enhanced Chemiluminescence Detection kit (RPN2108, Amersham Biosciences, Buckinghamshire, UK) and revealed using the ChemiDocTM XRS System (BioRad, Hercules, CA, USA).

Cell lysates from FHL124 cells were prepared using Daub’s lysis buffer supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF) and 10 μg/mL aprotinin for 20 min on ice and centrifuged at 16,060×g for 10 min. The protein content was determined by the BCA assay (Bio-Rad, Hemel Hempstead, UK) so that equal amounts of protein per sample were loaded onto 8% SDS–polyacrylamide gels and transferred to PVDF membrane using a semidry transfer cell. The membrane was blocked with PBS containing 5% nonfat dry milk and 0.1% Tween-20, hybridized with primary antibody (anti-LC3, (Sigma-Aldrich, Poole, Dorset); anti-ERK, anti-JNK, anti-p38, anti-β-actin (Cell Signaling Technology, Beverly, MA, USA), anti-EIF-2α, anti-BiP/GRP78 (BioSource International, Rockville, MD); anti-IRE1, anti-ATF6 (Abcam, Cambridge, UK)) followed by incubation with secondary antibody (Amersham Biosciences, Bucks, UK). Proteins were detected using the ECL plus blotting analysis system (Amersham Biosciences).

Immunoblotting was carried out as described previously^{5 (link)}. Proteins were separated on SDS-PAGE gels, electro-transferred onto polyvinylidene difluoride membranes and incubated overnight at 4 °C with the following antibodies: anti-Atg3, anti-Atg5 (D1G9), anti-Atg7 (D12B11), anti-Beclin (D40C5), anti-caspase-3 (Cell Signaling), anti-Bcl-2 (Millipore), anti-Bax (Millipore), anti-LC3 (Sigma-Aldrich), anti-LXRα (SantaCruz), anti-LXRβ (SantaCruz), anti-Nur77 (Active motif), anti-NOR1 (R&D systems), anti-PARP (Cell Signaling), anti-Vps34 (Cell Signaling), or anti-Actin (Millipore). For visualization, an ECL plus kit (Amersham Biosciences) was used, and chemiluminescence was measured by autoradiography (Supplementary Fig. 9). Specific bands were quantified with ImageQuant software.

GTCs were cultured in 24-well plates and infected with B.suis.S2 or B.suis.S2-mCherry for 24 h. Immunofluorescent staining of caspase-3, GRP78, CHOP, phosphoIRE1α, IRE1α, and LC3 was performed. The GTCs were fixed in 4% paraformaldehyde for 30 min and then permeabilized for 15 min with 0.1% Triton X-100 in PBS, subsequently blocked for 1 h with 5% BSA in PBS at room temperature, and co-incubated with anti-caspase-3 (Santa Cruz, 1:50 dilution), anti-CHOP (Santa Cruz, 1:50 dilution), anti-GRP78 (Santa Cruz, 1:50 dilution), anti-phosphoIRE1α (Abcam, 1:500 dilution), anti-IRE1α (Santa Cruz, 1:50 dilution), or anti-LC3 (Sigma, 1:500 dilution) antibodies at 37°C for 2 h. After washing and incubation with an anti-rabbit secondary antibody (for CHOP, phosphoIRE1α, IRE1α, LC3, and caspase-3) (Invitrogen, A21206; 1:500 dilution) or an anti-goat secondary antibody (for GRP78) (Invitrogen, A21432; 1:500 dilution) at 37°C for 1 h, the nuclei were stained with 4′, 6-diamidino-2-phenylindole (DAPI) for 3–5 min. The fluorescent signals were examined under a Nikon A1R si confocal microscope system.

The number of LC3 puncta and colocalization of LC3 with acidified lysosomes was determined by confocal microscopy as previously described^{61 (link)}. Briefly, BMM cultured on cover slips were infected with IOE at MOI of 5 or left uninfected. Cells were then washed 3X with PBS, fixed with 2% paraformaldehyde for 20 min, and permeabilized with 0.1% Triton X-100 in PBS for 30 min. After blocking with 5% BSA (Sigma-Aldrich, A2153) for 60 min, the primary antibodies; anti-LC3 (Sigma, 50 ug/mL) was added for 1 h at room temperature. Cells were washed and then incubated with fluorescent labeled anti-rabbit secondary antibody DyLight (VectaFluor, 1:500) for 1 h. Nuclei were stained with DAPI and cells were analyzed by confocal microscopy (Olympus Flouview 1000). Analysis of acidified lysosome was performed using LysoTracker Red (cat. L-12492, Thermofisher) at 37 °C for 1 h and assessed with a confocal microscope (Olympus Flouview 1000).