Protease and phosphate inhibitor cocktail

Liver tissues (n = 8) were lysed in RIPA buffer (50 mM Tris-HCl at pH 7.4, 150 mM sodium chloride, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate) with protease and phosphate inhibitor cocktail (Merck, Ashland, MA, United States) using a sonicator. Crude lysates were centrifuged at 20,000 × g for 20 min at 4°C. A part of the supernatant (n = 8) was used to measure the level of TNF-α, IL-1β, IL-6 and transforming growth factor (TGF)-β1 using commercial ELISA kits (eBioscience, San Diego, CA, United States). The absorbance at 450 nm was read by a microplate reader (Bio-Rad, Hemel Hempstead, United Kingdom).

hPDLSCs-OE-GRP78 were grown under normal growth and differentiation conditions with or without DMP1 treatment for 24 h. Cells were then harvested and lysed in RIPA buffer (cell signalling) containing protease and phosphate inhibitor cocktail (Millipore). Centrifugation was then performed at 11,500 ×g for 15 min at 4° C and the supernatants were used as total cellular proteins. Protein concentrations were measured using the Bio-Rad Protein Assay Dye Reagent (BIO-RAD) with BSA as standard. 25 μg of total proteins were loaded on a 10 % SDS-polyacrylamide gel. The proteins were transferred onto a nitrocellulose membrane following electrophoresis, blocked with 5 % skim milk (Merkel et al., 2019 (link)). The membranes were incubated with the following primary antibodies: anti-TRIP-1 rabbit polyclonal antibody (1/1000; Invitrogen), anti-FL DMP1 (1/1000; house-made), anti-DPP rabbit polyclonal antibody (1/1000; house-made) (Eapen et al., 2012 (link)). Anti-tubulin mouse monoclonal antibody (1/5000; Invitrogen) was used as a loading control. The blots were incubated in either anti-mouse or anti-rabbit secondary conjugated with HRP. Each of the blots were washed 4 times with PBS, and the bands were visualised using chemiluminescence detection (Thermo Fisher Scientific) using X-ray films according to the manufacturer’s protocol.

Alpha modification of Eagle's medium (AMEM), phosphate‐buffered saline (PBS), trypsin, and antibiotic–antimycotic preparations were purchased from Wisent (St Bruno, Quebec, Canada). Fetal bovine serum (FBS), horse serum (HS), Lipofectamine RNAiMAX, and Opti‐MEM 1X Reduced Serum Medium were purchased from Thermo Fisher Canada (Burlington, Ontario Canada). Amino acid‐free RPMI 1640 medium was purchased from US Biologicals (Salem MA). Sodium 4‐methyl‐2‐oxovalerate (sodium salt of KIC), 2‐deoxyglucose, protease and phosphate inhibitor cocktails, anti‐BCAT2 and anti‐gamma tubulin antibodies, siRNA oligonucleotides, amino acid standard, o‐Phthalaldehyde, interleukin‐6, and homocysteine were purchased from Sigma Aldrich (Oakville, Ontario, Canada). Phospho (ph) ribosomal protein S6 kinase 1 (S6K1) (T389), ph‐ribosomal protein S6 (S6) (S235/236), ph‐Akt (S473), ph‐SAPK/JNK (T183/Y185), ph‐glycogen synthase (S641), BCKDH‐E1α, horseradish peroxidase (HRP)‐conjugated anti‐rabbit and anti‐mouse secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA). [³H]‐2‐deoxyglucose was purchased from Perkin Elmer (Markham, Ontario, Canada) while chemiluminescence substrate was from Millipore (Etobicoke, Ontario, Canada). TNF‐α was purchased from Shenandoah Biotechnology (Warwick, PA).

Half-brain sections from 6-month-old mice were homogenized in a non-denaturing lysis buffer (50 mM Tris–HCl pH 7.4, 150 mM NaCl, 1% NP-40, 0.25% sodium deoxycholate, 0.1% SDS, 2 mM EDTA, 1 mM PMSF), supplemented with protease and phosphate inhibitor cocktails (Sigma-Aldrich cat# 4693132001 and 4906837001). The homogenates were cleared by centrifugation at 13,000 × g at 4°C for 25 min, and the protein concentration in the collected supernatant was measured using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Protein extracts (20 μg of protein for each sample), were incubated in a boiling bath for 10 min and analyzed by SDS-PAGE on 4–20% precast polyacrylamide gradient gel (Bio-Rad, 4561096). Western blot analyses were performed according to standard protocols using antibodies against MBP (1:1000, Abcam, cat# ab218011), MAG (1:1000, Abcam, cat# ab89780), and α-tubulin (1:2000, DSHB, cat# 12G10) as a control. The immunoblots were revealed by chemiluminescence with SuperSignalWest Pico PLUS (Thermo Fisher Scientific, Waltham, MA, USA). Detected bands were quantified using ImageJ 1.50i software (National Institutes of Health, Bethesda, MD, USA) and normalized for the intensity of the α-tubulin band.