Anti grp78

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.

Lung homogenates or cell lysates were subjected to denaturating SDS-PAGE, followed by electroblotting and immunoblotting for anti-ATF4, anti-GRP78, anti-CHOP (Santa Cruz Biotechnology, Dallas, TX), anti-ATF6α, anti-IRE1 (Enzo Life Sciences, Farmingdale, USA), anti-XBP-1 (Novus Biologicals, Littleton, CO), anti-eIF2α, anti-phospho eIF2α, anti-phospho PERK, anti-PERK, anti-phospho AKT(Thr308), anti-phospho p70S6K (Cell Signaling Technology, Danvers, MA) or anti-phospho IRE1 (Abcam, Cambridge, MA). Blots were developed using corresponding HRP-conjugated secondary antibodies and detected using a chemiluminescent system (Amersham ECL Plus; GE Healthcare, Piscataway, NJ). Band intensities were quantified with a LAS-1000 plus system (Fuji Film, Japan).

Five rats of each group were decapitated, and the whole brains were removed and immediately placed in an ice-cold dish. Then the mPFC was dissected according to the atlas [32 ] by use of a stereomicroscope and was quickly frozen in liquid nitrogen and stored-80°C. The mPFC was homogenized with a sample buffer containing 200 mM TBS, pH 7.5, 4% SDS, 20% glycerol, 10% 2-mercaptoethanol and denatured by boiling for 5 min. The protein fraction (50 μg/lane) prepared from each sample was separated by 8% (w/v) gradient sodium dodecyl sulfate(SDS)-polyacrylamide gel electrophoresis (PAGE) and electroblotted to a PVDF membrane (Millipore, Bedford, MA, USA) from the gel by a semi-dry blotting apparatus (Bio-Rad Laboratories, Inc., Hercules, CA, USA). After blocked with 5% dried skim milk, the membrane was incubated with I antibody (anti-GRP78, Santa Cruz, USA, 1:1000; anti-ERP57, Santa Cruz, USA, 1:1000; anti-GADPH, Zhongshan Glodenbridge Biotechnology, China; 1:1000) for 24 h at 4°C and II antibody ( Boster Biological Technology Ltd., 1:3000) for 2 h at room temperature. Blots were subjected to autoradiography (ECL reagents, Amersham Pharmacia Biotech, Buckinghamshire, UK). The optical density (OD) was analyzed on the Gel Image Analysis System. The expression of GRP78 and ERP57 were determined by calculating the OD ratio of GRP78/GADPH and ERP57/GADPH.

Anti-GRP78, PERK, phosphor-PERK, JAK2, and STAT3 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). We purchased thapsigargin from Sigma (USA) and GSK2606414 and AG490 from Selleck Chemicals (USA). TGF-β1 and FN ELISA kits were bought from Boster (Wuhan, China), a collagen I ELISA kit was from BlueGene Biotech. (Shanghai, China), and a cell counting kit (CCK-8) was bought from Dojindo (Kumamoto, Japan). Rat renal proximal tubule epithelial cells (NRK-52E) were obtained from the American Type Culture Collection.

Liver homogenates and cells were lysed in ice-cold RIPA buffer (50 mM Tris HCl (pH 7.4), 250 mM NaCl, 1% Nonidet P-40, and protease inhibitor cocktail). The protein samples (50 µg) were separated by 4%–12% sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred onto PVDF membranes. After blocking with 5% BSA or 5% non-fat dry milk, the membranes were incubated overnight with the following primary antibodies: anti-p-mTOR, anti-mTOR, anti-p-p70s6kinase, anti-p70s6kinase, anti-p-4EBP-1, anti-4EBP-1, anti-CHOP, anti-p-eIF2α, and anti-eIF2α (Cell Signaling Technology, Boston, MA, USA), anti-ubiquitin, anti-GRP78, anti-p-PERK, anti-PERK, anti-Bax, anti-lamp-1, anti-Tom20, anti-cathepsin B, and β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-p62 (SQSTM1; MBL International, Woburn, MA, USA), and anti-LC3II (Novus Biologicals, Littleton, CO, USA). Immunoreactive bands were visualized using the ECL Western blotting protocol (Bio-Rad). Finally, the membranes were exposed to imaging film (Kodak BioFlexEcono Scientific Supplies, Citrus Heights, CA, USA), after which the film was developed using a Kodak X-OMAT 1000A Processor. Densitometric analysis of the bands was conducted using the Image J software (NIH, Bethesda, MD, USA).

Chemicals used were: bortezomib (LC Laboratories), Propidium Iodide and N-acetylcysteine (Sigma Aldrich). Primary antibodies used were: anti-Gas2^{36 (link)} anti-actin (Sigma/Aldrich) anti-USP1^{37 (link)} anti-USP33 (Millipore) anti-AKT (Cell Signaling) anti-UCH-L5 (Abcam), anti-USP18^{38 (link)} anti-HDAC4^{39 (link)} anti-MEF2D (BD Bioscience), anti-GRP78, anti-USP14, anti-Cofilin-1, anti-pCofilin-1 (Santa Cruz Biotechnologies).

Whole cell lysates were prepared and analyzed by Western blotting as described previously [32 (link)]. Proteins in cell lysates were separated by precast 8-20% SDS-polyacrylamide gel electrophoresis, and then electrophoretically transferred from the gel onto polyvinylidene difluoride membranes. After blocking, blots were incubated with anti-GRP78, anti-GRP94, anti-phospho-PERK, anti-caspase-12, anti-caspase-7, anti-CHOP, anti-PARP, and anti-β-actin antibodies (Santa Cruz Biotechnology) and anti-eIF2α (Cell Signaling Technology, Danvers, MA, USA) in PBS within 0.1% Tween 20 for 1 h followed by two 15 min washes in PBS with 0.1% Tween 20. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies for 60 min. Detection was performed with Western blotting reagent ECL (Amersham-GE Healthcare Life Sciences, Pittsburgh, PA, USA), and chemiluminescence was exposed by the Kodak X-Omat films.