Odyssey bioimager

At 21 weeks of age, LV tissue was collected at the end of working heart experiments, snap frozen and stored at −80°C until further analysis. Frozen tissues were homogenized in lysis buffer [in mmol/L: 20 Tris (pH 7.4), 5 EDTA, 10 Na₄O₇P₂ sodium pyrophosphate tetrabasic, 100 sodium, 9 fluoride, and 1% NP‐40] containing protease (Protease Inhibitor Cocktail (1× Halt TM 200 protease inhibitor, 201 Thermo scientific) and 1 mmol/L PMSF, Fluka Biochemika) and phosphatase inhibitors (2 mmol/L Sodium Orthovanadate, Sigma). The protein concentration of the lysate was determined using a bicinchoninic acid assay (Pierce). A total of 100 μg of protein was loaded and separated by SDS‐PAGE on a 7.5% or 10% polyacramide gel and transferred to a nitrocellulose membrane. The membrane was incubated with 50% blocking reagent for 1 h at room temperature. After washing with phosphate‐buffered saline solution, the membrane was incubated overnight at 4°C with primary antibodies for p‐AMPK (1:2000, Cell Signaling), SOD1 (1:1000, Santa Cruz Biochemicals), SOD2 (1:1000, Santa Cruz Biochemicals), Catalase (1:1000, Santa Cruz Biochemicals) or β‐actin (1:1000; Santa Cruz Biochemicals). The membrane was incubated with secondary antibody conjugated with fluorescent tag and blots were visualized with Li‐cor Odyssey Bioimager and quantified by densitometry with Odyssey V3.0 software (Li‐cor Biosciences).

Immunoblotting was performed according to our recent report [29 (link)]. Briefly, HEK293T cells were grown in 6-well tissue culture plates until they reached ~80% confluency. They were then treated with 50 μM IRW or its analogs for 24 h. Following 24 h incubation, the culture medium was carefully removed, and the cells were lysed in RIPA buffer. These cell lysates were run on sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE), transferred to nitrocellulose membranes, blocked in tris buffered saline with 5% low-fat milk (TPBS) solution, and immunoblotted with primary antibodies in 1:500 concentration. Next, after incubating overnight with primary antibodies, blots were washed with TPBS and incubated with the appropriate secondary antibodies. Finally, after washing excess secondary off with TPBS, the antibody reactive protein bands were detected using a Licor Odyssey BioImager (Licor Biosciences, Lincoln, NB, USA) and quantified by densitometry using Image Studio Lite 5.2 software (Licor Biosciences, Lincoln, NB, USA).

The cells were treated with varying concentrations of phosvitin, PPP or 0.5 mg/mL lactoferrin for 72 h. At the end of the incubation period, the culture medium was removed and the cells lysed in boiling hot Laemmle’s buffer containing 50 μM dithiothreitol (DTT, a reducing agent) and 0.2% Triton-X-100 to prepare samples for western blot, as described before [25 (link)]. These cell lysates were run in SDS-PAGE, blotted to nitrocellulose membranes and immunoblotted with antibodies against alkaline phosphatase (ALP; mouse monoclonal antibody from Santa Cruz Biotechnologies, Santa Cruz, CA, USA), RANKL (rabbit polyclonal antibody from Santa Cruz Biotechnologies) and the loading control α-tubulin (rabbit polyclonal antibody from Abcam, Cambridge, MA, USA). Anti-tubulin was used at 0.4 μg/mL, while the others were used at 1 μg/mL. Goat anti-rabbit and Donkey anti-mouse fluorochrome-conjugated secondary antibodies were purchased from Licor Biosciences (Lincoln, NB, USA). The protein bands were detected by a Licor Odyssey BioImager and quantified by densitometry using corresponding software (Licor Biosciences). Each band of ALP or RANKL was normalized to its corresponding band of loading control. Cell lysates from untreated cells were loaded onto every gel. The results were expressed as percentages of the corresponding untreated control results.

The cells were seeded on 48-well tissue culture plates at a concentration of 1 × 10⁴ cells/well, and cultured in DMEM with 10% FBS for 6 h to allow the cells to attach to the surface. Then, RAW 264.7 cells were pretreated with ovotransferrin (1–1000 μg/mL) for 2 h prior to stimulation with RANKL (100 ng/mL) and incubated together for 12 h. After removing the culture medium, cells were lysed in boiling hot Laemmle’s buffer with 50 μM dithiothreitol (DTT) and 0.2% Triton-X-100 and used for Western blot analysis as described previously [23 (link)]. These cell lysates were run in SDS (sodium dodecyl sulfate)-PAGE (polyacrylamide gel electrophoresis), blotted to nitrocellulose membranes, and immunoblotted with different antibodies described in Section 2.1. The dilution factor was 1:1000 for most of the antibodies, except for phosphor-p44/42 MAPK (1:2000), Cathepsin K (1:2000), TRAF6 (1:200), NFATc1 (1:200) and α-Tubulin (1:100,000). All antibodies were diluted into phosphate buffer saline (PBS) containing 5% tween 20. The α-tubulin was used as the internal reference. The protein bands were detected using a Licor Odyssey BioImager and quantified by densitometry using the corresponding software (Licor Biosciences, Lincoln, NB, USA). The results are expressed as percentage of the corresponding untreated control.

The confluent A7r5 cells were placed in a quiescing medium (the same as that of the growth medium but with 1% FBS). A7r5 cells were treated with 2.5 mg/mL of SPHs for 24 h for ACE2 detection. EA.hy926 cells were treated with 2.5 mg/mL of SPHs for 18 h prior to the addition of 10 ng/mL of tumor necrosis factor-alpha (TNFα) for a 6 h co-treatment for detection of intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). The dose of hydrolysate was selected based on our previous studies [9 (link)]. Cells were lysed in boiling Laemmle’s buffer containing 50 mM Dithiothreitol (DTT) and 0.2% Triton-X-100.
Cell lysates were loaded onto a 9% separating gel and transferred to a nitrocellulose membrane (diameter 0.45 µm, 1620115, Bio-Rad, Montreal, QC, Canada) for incubation with antibodies. Bands of ACE2 (ab87436, Abcam, Toronto, ON, Canada), ICAM-1 (sc-8439, Santa Cruz, Dallas, TX, USA) and VCAM-1 (sc-8304, Santa Cruz, Dallas, TX, USA) were normalized to α-tubulin (ab15246, Abcam). Donkey-anti-rabbit 800 CW or donkey-anti-mouse IRDye 680 RD secondary antibodies (Licor Biosciences, Lincoln, NE, USA) were used to visualize the fluorescent bands in a Licor Odyssey BioImager, which were quantified using Image Studio Lite 5.2 (Licor Biosciences).

The cells were seeded on 48 well tissue culture plates at a concentration of 1 × 10⁴ cells/well and incubated in α-MEM with 10% FBS. The cells were treated with IRW (50 μM and 25 μM) and 1 μM of Ang II. After incubation, the culture medium was removed, and the cells lysed in boiling Laemmle’s buffer containing 50 μM dithiothreitol (DTT) and 0.2% Triton-X-100 to prepare samples for Western blot as described previously. These cell lysates were run in SDS-PAGE, blotted to nitrocellulose membranes, and immunoblotted with specific antibodies. The protein bands were detected by a Licor Odyssey BioImager and quantified by densitometry using corresponding software (Licor Biosciences, Lincoln, NB, USA). Each band was normalized to its corresponding band of loading control. Cell lysates from untreated cells were loaded onto every gel. The results were expressed as a percentage of the corresponding untreated control.