Chemiluminescence detection

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.

Skeletal muscle and liver lysates were prepared from powdered tissue by homogenizing in 25 mM HEPES, pH 7.4, 1% Igepal CA630, 137 mM NaCl, 1 mM PMSF, 10 μg/mL aprotinin, 1 μg/mL pepstatin, 5 μg/mL leupeptin, 10 mM Na₄P₂O₇, 100 mM NaF, and 2 mM NaVO₄ using a Sonifier 450 homogenizer (VWR, Radnor, PA, USA). The samples were centrifuged at 12,000× g for 10 min at 4 °C. Protein concentrations were determined using a BCA assay (Thermo Fisher Scientific, Rockford, IL, USA) according to the manufacturer’s instructions. Tissue supernatants (50 μg) were resolved by SDS-PAGE and subjected to immunoblotting using chemiluminescence detection (Thermo Fisher Scientific, Rockford, IL, USA) and quantified using Image J software after normalizing to β-actin or when appropriate, total protein kinase B (AKT) or total AMP kinase alpha subunit AMPKα. Nitrocellulose membranes were incubated with antibodies for 1–2 h at room temperature or overnight at 4 °C as indicated (Table S2).

bEnd.3 cells were washed with PBS and lysed with radioimmunoprecipitation assay buffer containing cocktail protease inhibitors (Bio-Rad, Hercules, CA, United States) and boiled with Laemmli buffer and 10% 2-mercaptoethanol (Bio-Rad, Hercules, CA, United States). Blots were probed with anti-transferrin receptor (TfR) antibody (1:1,000 dilution; Thermo Fisher Scientific, Waltham, MA, United States) or anti-ferroportin antibody (1:1,000 dilution; Novus Biologicals, Littleton, CO, United States) overnight at 4°C. Membranes were exposed to the appropriate horseradish peroxidase–conjugated secondary antibodies, followed by chemiluminescence detection (Thermo Fisher Scientific, Waltham, MA, United States). Equal protein loading was controlled by re-probing the membrane with anti–β-actin antibody (1:1,000 dilution; Santa Cruz Biotechnology, Dallas, TX, United States). Chemiluminescence was detected using the UVP ChemiDoc-It TS2 Imager (Upland, CA, United States), and Image J (NIH, Bethesda, MD, United States) was used for Western blot signal quantification.

In order to reduce the number of animals used in the present study, 1 mg/kg D. salina was used as the effective dose to investigate the mechanism underlying the cardioprotective effect of D. salina. Protein expression was evaluated by western blot as previously described procedures [44 (link)]. The primary antibodies against COX-2 (Cayman Chemical, Ann Arbor, MI, USA) included TLR4, phospho-JAK2 (p-JAK2), JAK2 (Santa Cruz, Dallas, TX, USA), phospho-NF-κB (p-NF-κB), NF-κB, phospho-IκB (p-IκB), IκB, procaspase-3, cleaved caspase-3 (Cell Signaling, Danvers, MA, USA), signal transducer and activator of transcription-1 (STAT1), phospho-STAT1 (p-STAT1) (Abcam, Cambridge, UK), light chain 3 (LC3), p62 and Beclin-1 (Novus, Centennial, CO, USA). β-actin (Abcam, Cambridge, UK) was used as an internal loading control. The membranes were incubated with an HRP-conjugated secondary antibody (Jackson Immuno Research Laboratories, West Grove, PA, USA) prior to chemiluminescence detection (Thermo Scientific, Waltham, MA, USA).

Western blotting was performed as previously described in ref. ^{45 (link)}. The primary antibodies included CUL4B (Sigma, C9995), RUNX2 (CST, 12556 S), OSTERIX (Abcam, ab209484), PPARγ (CST, 2435 S), C/EBPα (CST, 8178 T), KLF4 (CST, 4038 T), C/EBPδ (CST, 2318 T) and GAPDH (Abmart, M20006S). These antibodies were used at a 1:1 000 dilution. Secondary antibodies included anti-rabbit or anti-mouse horseradish peroxidase (HRP) (Abmart). The membranes were subjected to chemiluminescence detection (Thermo) and then exposed by an Amersham Imager 600 (GE) to visualize the bands. Estimates of protein amounts were obtained by measuring the area of protein bands using ImageJ and were normalized to GAPDH protein amount in the respective samples.

The cells were lysed in buffer containing 62.5 mM Tris-Cl pH 6.8, 10% glycerol and 2% SDS. Thirty micrograms of the samples were separated by 9.5–20% gradient SDS-PAGE and subjected to Western blotting. Anti-S100A8, anti-S100A9, mouse monoclonal anti-actin (clone AC-15, Sigma-Aldrich, St. Louis, MO, United States) as well as horseradish peroxidase-labeled goat anti-rabbit and rabbit anti-mouse (both Sigma-Aldrich) antibodies were used followed by chemiluminescence detection (Thermo Fisher Scientific). ChemiDoc XRS⁺ Molecular Imager and Quantity One analysis software (both Bio-Rad, Philadelphia, PA, United States) were used for quantification. Interleukin-8 (IL-8, CXCL8), Epithelial-derived Neutrophil-Activating peptide 78 (ENA-78, CXCL5), Neutrophil Activating Protein-2 (NAP-2, CXCL7) and Growth-Regulated Oncogene-α (GRO-α, CXCL1) concentrations were measured with DuoSet (R&D Systems, South Beloit, IL, United States) according to the manufacturer’s protocols.

Cells or brain tissues were lysed in 300 μl of lysis buffer [150 mM sodium chloride, 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulphate (SDS), 50 mM Tris-HCl (pH 7.5), 2 mM EDTA] containing mixture of Halt^TM protease and phosphatase inhibitors (1×) (Thermo Fisher Scientific). The brain tissues were individually homogenized and then centrifuged at 13,400×g at 4°C for 15 min. Protein concentration was determined using the Pierce BCA protein assay kit (Thermo Fisher Scientific). Bovine serum albumin was used as the standard. Proteins (20–30 μg) for each sample were separated using 12% sodium dodecyl sulfate-PAGE and transferred to polyvinylidene fluoride filter membranes (Bio-Rad) by the semi-dry electroblotting method. The membranes were blocked with 5% skim milk and incubated sequentially with the following primary antibodies against either c-Abl (rabbit monoclonal antibody, 1:1000; Santa Cruz), p-p65, p-65, p-IκB, IκB (rabbit monoclonal antibody, 1:1000; Cell Signaling), TNF-α (rat anti-mouse monoclonal antibody, 1:500; Millipore) or α-tubulin (mouse monoclonal antibody, 1:2000; Sigma-Aldrich) and horseradish peroxidase-conjugated secondary antibodies (anti-rabbit or mouse IgG antibody; Cell Signaling), followed by chemiluminescence detection (Thermo Fisher Scientific).