X omat 1000a processor

Liver tissue homogenates were prepared by homogenizing the tissue in lysis buffer (20 mM Tris, pH 7.4; 150 mM NaCl; and 0.5% Triton X-100) or RIPA buffer (150 mM NaCl; 50 mM Tris, pH 8.0; 2 mM EDTA; 1% Nonidet P-40; and 0.1% SDS) supplemented with a protease inhibitory cocktail tablet (Roche, Indianapolis, IN, USA) and phosphatase inhibitory cocktails 2 and 3 (Sigma-Aldrich). Lysates were cleared by centrifugation and analyzed by gel electrophoresis. Protein concentration was determined via Bradford protein assay (Bio-Rad, Hercules, CA, USA) using BSA as a standard, and verified by Coomassie blue gel staining. Lysates (45 μg) were resolved by SDS-PAGE (Bio-Rad) and then transferred to nitrocellulose membranes. Membranes were blocked for 1 h with 5% skim milk in Tris-buffered saline (0.137 M NaCl, 0.025 M Tris, pH 7.4) containing 0.1% Tween-20 (T-TBS). Antibodies were diluted according to the manufacturers’ recommended protocols. Protein signals were visualized using an enhanced chemiluminescence (ECL) reagent (SuperDetect^TM ECL Western Blotting Detection Reagent, DaeMyung Science Co., Ltd, Seoul, Korea). Finally, membranes were exposed to imaging film (Kodak BioFlexEcono Scientific Supplies, Citrus Heights, CA, USA), which was developed using a Kodak X-OMAT 1000A Processor.

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).

After the stimulation/transfection (transfection with lipofectamine 2000 (Thermo Fisher Scientific, catalog # 11668019)) of PMA-differentiated THP1 dual reporter cells, or J774 dual reporter cells in 24-well plates, the cells were washed with ice-cold PBS and directly lysed in 150 µl of sample loading buffer containing 0.1 M Tris/HCl (pH 6.7), 2% SDS, 10% glycerol, bromophenol blue, and 1% β-mercaptoethanol. Lysates were boiled at 95°C for 10 min and vortexed well before loading onto 10% SDS-PAGE gels. Proteins were transferred to PVDF membranes using an iBlot2 gel transfer device (Thermo Fisher Scientific, Massachusetts, USA). Next, the membranes were blocked in TBST containing 5% skim milk at room temperature for 1 h and incubated with the appropriate primary antibodies diluted in TBST containing 5% BSA at room temperature for 1 h. The membranes were then washed three times with TBST and incubated with HRP-linked anti-rabbit IgG antibody (Cell Signaling Technology, catalog # 7074) in TBST containing 5% BSA for 1 h at room temperature. Protein bands were visualized by treating the membrane with Immobilon Western Chemiluminescent HRP Substrate (Merck Millipore, Darmstadt, Germany, catalog # 42029053) and developing on the films using a KODAK X-OMAT 1000A Processor.