Anti cathepsin b

Stimulated cells and tissue samples were harvested for protein extraction and western blotting analysis with standard protocols as previously described^{12 (link),56 (link)}. Cytoplasmic fractionations were performed with NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific, Rockford, IL, USA) according to the manufacturer’ s protocol. Briefly, cells were harvested, washed and pelleted. Then added ice-cold CER I to the cell pellet and incubated the tube for 10 min on the ice. After that, added ice-cold CER II to the tube and incubated for 1 min on the ice. The volume ratios of cell pellets, CER I and CER II are 10: 100: 5.5. At last, centrifuged the tube for 5 min at 16, 000 × g and transferred the supernatant to a clean pre-chilled tube. Stored this tube in −80 °C until use. The following antibodies were used: anti-GAPDH (1:10000; Genetex, Irvine, CA, USA); anti-DAPK1, anti-MLC (1:1000; Abcam, Cambridge, MA, USA); anti-p-MLC (1:500; Cell Signaling Technology, MA, USA); anti-IL-1β (1: 2000; R&D Systems, Wiesbaden, Germany); anti-NLRP3, anti-caspase-1, anti-ASC (1:1000; Adipogen, San Diego, CA, USA)]; anti-cathepsin B (1:300; Santa Cruz Biotechnology, Santa Cruz, CA, USA); secondary antibodies (1:400; Jackson ImmunoResearch Laboratories, PA, USA). The intensity of protein bands was analyzed with the Image J software (NIH) and normalized to GAPDH.

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).

The following antibodies were purchased from the indicated sources: anti-EGFR (A-10), Santa Cruz Biotechnology (catalog no. sc-373746); anti-GBA, Abcam (catalog no. ab55080); anti-GAPDH (14C10), Cell Signaling Technology (catalog no. 3683); anti-cathepsin B, Santa Cruz Biotechnology (catalog no. sc-365558).