Proteinase and phosphatase inhibitor

Total protein was extracted from heart tissues using a RIPA buffer containing proteinase and phosphatase inhibitor (Roche; Germany). After determining the protein concentrations using a BCA kit (Thermo; United States), protein lysates were separated by SDS-PAGE and transferred to PVDF membranes (Millipore; United States). The membranes were incubated with the corresponding primary antibodies, such as ERK1/2, phosphor-ERK1/2, MMP9, Nrf2 and GAPDH (Abcam; United States) overnight at 4°C before being incubated with goat anti-rabbit lgG (Abcam; United States). The western blot bands were detected using ECL kit (Keygene; China). For quantification by imageJ (NIH; United States), the specific protein expression levels were normalized to GAPDH levels.

Protein extraction was the first step before any additional procedure. Initially, samples were embedded in cold RIPA buffer (Sigma Aldrich) supplemented with proteinase and phosphatase inhibitor (Roche, Basel, Switzerland) and later sonicated for 10 s for membrane disruption. Samples were then centrifuged for 10 min at 10,000 rpm to isolate the total proteins that remained in the supernatant.
Protein concentration was calculated using Pierce™ BCA Protein Assay Kit (Thermo Fischer Scientific). As recommended by the manufacturer, BSA standard dilutions were carefully made in RIPA buffer in a working range of 2000–20 µg/ml. Samples were then incubated with supplied reagent at 37 ℃ for 30 min. Later, 562 nm wavelength was measured in a Benchmark Plus microplate reader (Bio-Rad, Hercules, CA, USA). Samples were diluted to a final concentration of 2 µg/µl with RIPA buffer.

Tissues or cells were homogenized in 1× RIPA lysis buffer (Millipore, 20-188) with proteinase and phosphatase inhibitors (Roche, 04693124001, 04906845001), and the supernatant was collected after centrifugation at 14,000 × g for 10 min. Protein quantification was performed using BCA protein assay (Pierce, 23227). Total protein for all samples were separated on 4–20% Criterion^TM XT Bis-Tris gels (Bio-Rad), transferred to nitrocellulose membrane (Bio-Rad), and stained with Revert^TM 700 Total Protein Stain Kit (LI-COR, 926-11016) to verify protein concentration and loading accuracy. After blocking with Odyssey blocking buffer (LI-COR, 927-70001), the membrane was incubated with a goat anti mouse/human myeloperoxidase (MPO) antibody (R&D, AF3667), a rabbit anti-mouse phospho (p)-p65 nuclear factor (NF)-κB (CST, 3033S), or a rabbit anti-mouse p65 NF-κB (CST, 8242S) overnight at 4°C, followed by incubation with a donkey anti-goat or anti-rabbit 680RD secondary antibody (LI-COR, 925-68074 or 926-68073) for 45 min at room temperature. The signal was measured at the wavelength of 700 nm with the LI-COR imaging system (Odyssey CLx). The signal of total protein was used as the internal loading control for each lane, and data were quantified as the ratio of MPO to total protein signal (12 (link)). The ratio of p-p65 to p65 was quantified to represent NF-κB activation.

Pulverized 3D-LTCs were lysed in T-PER lysis buffer (Thermo Fisher Scientific, Germany) containing proteinase and phosphatase inhibitors (Roche, Switzerland). Protein concentration was assessed using the BCA assay (Thermo Fisher Scientific, Germany) according to the manufacturer’s instructions. 15 μg of total protein was separated on SDS-polyacrylamide gels and transferred to PVDF membranes (Biorad, USA). The membranes were blocked in 5% nonfat dry milk (Applichem, Germany) and incubated with the primary antibody (at 4 °C overnight followed by 1 h at RT). Subsequently the blots were incubated with respective secondary, HRP-conjugated, antibody (GE-Healthcare) for 1 h, washed and visualized using chemiluminescence reagents (Pierce ECL, Thermo Fisher Scientific, Germany) with the ChemiDocTMXRS+ system. Analysis of secreted collagen was performed by concentrating 200 μl of supernatant from the same number of 4-mm punches generated from 3D-LTCs in each group using Nanosep 10 K OMEGA columns (Pall Corporation, MI, USA) followed by dilution in 60 μl lysis buffer and as previously described [25 (link)].

In Western Blot analysis, whole cell lysate was prepared from primary culture of macrophages treated with indicated stimuli in the Results or Figure Legends by RIPA buffer (Sigma Aldrich, St. Louis, MO) supplemented with proteinase and phosphatase inhibitors (Roche, Indianapolis, IN). After Sodium Dodecyl Sulfate- Polyacrylamide gel Electrophoresis (SDS-PAGE), separated proteins were transferred to a PVDF membrane (Immobilon-P, Merck) and blocked with an ECL plus blocking agent (GE healthcare, Buckinghamshire, UK). Blotted membranes were, then, incubated with primary antibodies followed by incubation with anti-IgG antibody conjugated with horseradish peroxidase (GE healthcare). Finally, the signal was detected by the chemiluminescent reagent (ECL Prime Western Blotting Detection System, GE healthcare). α-Tubulin was served as an internal control.
Primary antibodies used in this study are as follows: rabbit monoclonal anti-p65 antibody (Cell Signaling Technology, Danvers, MA), rabbit monoclonal anti-phospho NF-κB p65 (ser536) antibody (Cell Signaling Technology), mouse monoclonal anti-IκBα antibody (Cell Signaling Technology), mouse monoclonal anti-α-tubulin antibody (Sigma Aldrich).

Spleen samples were homogenized in RIPA buffer (Santa Cruz Biotechnology, CA) with proteinase and phosphatase inhibitors (Roche, Indianapolis, IN). Proteins were separated by SDS-PAGE, transferred to a PVDF membrane (Millipore, Billerica, MA), incubated with anti-Ezh2 antibody (Cell Signaling Technology, Boston, MA), and subsequently with secondary anti-rabbit IgG antibodies labeled with HRP, both in blocking buffer (1% dry milk). Proteins were visualized with enhanced chemiluminescence substrate (Fisher Scientific, Hampton, NH).