Nonfat milk powder

Cell proteins were extracted using ice-cold radio-immunoprecipitation assay buffer (RIPA, Cell Signaling Technology, 9806) with complete protease inhibitor mixture (Roche Applied Science, 04693124001). Protein was separated by gel electrophoresis in 10–15% SDS-polyacrylamide gels and subsequently transferred onto polyvinylidene difluoride (PVDF) membranes (Bio-Rad, 1704156). Following blocking with TBST (Tris-buffered saline with 0.1% Tween-20) buffer containing 5% (w:v) nonfat milk powder (Bio-Rad, 1706404), the blots were probed with the corresponding primary antibodies and secondary antibody. Blots were visualized using the Pierce ECL kit (Pierce, 32106) and ChemiDoc MP Imaging System (Bio-Rad, 12003154).

Cell pellets were lysed on ice in RIPA buffer (Pierce, Rockford, IL) supplemented with Complete Mini EDTA-free Protease Inhibitor Cocktail (Roche, Indianapolis, IN) and Phosphatase Inhibitor Cocktail (Sigma, St. Louis, MO). Protein concentrations were determined by Bradford assay (Bio-Rad, Hercules, CA). Protein(50ug) was diluted 1:5 in 5X protein loading buffer (Fermentas, Glen Burnie, MD), boiled at 80°C for 5 minutes, electrophoresed on a 4-20% Tris-Glycine gel, and transferred using a Trans-Blot Turbo Transfer System (Bio-Rad, Hercules, CA). Membranes were blocked in 5% Non-fat milk powder (BioRad), incubated with primary antibody overnight at 4°C, incubated with HRP-coupled secondary antibody 1 hour at room temperature, developed with Visualizer Western Blot Detection Kit (Millipore, Billerica, MA), and visualized on a LAS-4000 imager (Fujifilm, Edison, NJ). The following antibodies were used at 1:1000 dilutions: Rabbit anti-FOXM1 (#5436), PLK1(#4513), pCDC2(#9111), CDC2 (#9112), MRE11, Survivin (#4895), mouse anti-STAT3(#9139), pSTAT3 (#9138), ß-Actin (#3700) (Cell Signaling Technology., MA); mouse-anti -pγH2AX (#05636, Millipore., MA), rabbit-anti RAD51 (sc-8348, Santacruz., CA), mouse anti-53BP1 (#612522, BD Transduction Laboratories., CA). Secondary antibodies, goat anti-rabbit-HRP, goat anti-mouse-HRP (Santa Cruz, CA) were used at 1: 10,000 dilution.

LV infarct tissues were weighted and placed in their respective 1.5 mL tubes with 16 μl of 1xPBS (without calcium, Invitrogen) and 1x proteinase inhibitor (Roche Diagnostics). While at 4°C, the samples were dis-membrated in short, 10 second intervals using a sonic dismembrator until homogenous, then centrifuged at maximum speed (14,000 rpm). The supernatant was used as the soluble protein fraction. The kidney protein was extracted using RIPA buffer. Total protein determined with Bradford assay. Electrophoresis of 10 μg of protein from each tissue with XT bis-tris-4-12% gel (Bio-Rad Inc.) in MOPS buffer (Bio-Rad) was performed. Samples were then transferred onto nitrocellulose membrane (Bio-Rad Inc.) and a total protein stain was performed with Pierce reversible protein stain, nitrocellulose membrane kit (Thermo Scientific Inc.). Each membrane was blocked for 1 hour at room temperature with 5% non-fat milk powder (Bio-Rad) dissolved in PBS-T and probed with primary antibodies. Primary antibodies (FPR2/ALX 1:1000; Ccl2 1:5000; arg-1 1:5000, GPR40 1:1000; 5LOX 1:1000) were probed overnight at 4°C, subsequently washed with PBS-T, and a secondary antibody was applied (Biorad). Proteins were detected using Femto chemiluminescence detection system. Densitometry of protein blots were assessed with ImageJ software (NIH, USA).

IPSCs were lysed in radio-immunoprecipitation assay (RIPA) buffer, pH 8.0, containing phosphatase and protease inhibitors (Sigma-Aldrich). Lysates were kept on ice for 30 min and centrifuged at 16,000× g for 20 min at 4 °C. Samples containing an equal amount of total proteins (20 μg) were resolved by 7.5% sodium dodecyl sulfate (SDS)-polyacrylamide gel (Biorad, Hercules, CA, USA). Proteins were transferred to nitrocellulose membrane using a dry transfer system (Biorad), and blots were blocked with 5% non-fat milk powder (Biorad) in Phosphate-buffered saline (PBS) containing 0.05% Tween-20 for 1 h at 4 °C and incubated with mouse monoclonal anti-TBCD (1:500, ThermoFisher), mouse monoclonal anti-GAPDH (1:1000, Santa Cruz, CA, USA) and anti-mouse HRP-conjugated secondary antibody (1:3000, ThermoFisher). Immunoreactive proteins were detected by an enhanced chemiluminescence (ECL) detection kit (ThermoFisher) according to the manufacturer’s instructions, and an Alliance Mini HD9 (Uvitec) was used for chemiluminescence detection.

Liver tissue and HepG2 cells were lysed for 20 min on ice in a radioimmunoprecipitation assay buffer containing a complete protease inhibitor cocktail (Roche), followed by centrifugation at 18,000×g for 15 min at 4 °C. The total protein concentration was measured using a bicinchoninic acid (BCA) protein assay (Thermo Fisher Scientific, Waltham, MA, USA). Lysates were boiled in 1× SDS Laemmli sample buffer for 5 min, separated by SDS-PAGE, and transferred to polyvinylidene fluoride membranes (Merck Millipore, Darmstadt, Germany), which were subsequently blocked with 5% nonfat milk powder (Bio-Rad, Hercules, CA, USA). The blots were probed with primary antibodies against ubiquitin, p62, phospho-CaMKII, CaMKII, NLRP3, caspase-1, TXNIP, α-tubulin, β-actin, and GAPDH. The blots were then washed and incubated with the appropriate horseradish peroxidase-conjugated secondary antibodies. After washing, the protein bands were detected by enhanced chemiluminescence using the SuperSignal West Femto chemiluminescent substrate (Thermo Scientific, Rockford, IL, USA). ImageJ software (National Institutes of Health, USA) was used to calculate the protein band intensities.

mMSCs were grown in normal or osteogenic medium with Zn-CS/β-GP or Zn-CS/β-GP/nHAp hydrogels for different time periods. The cells were then washed with cold phosphate-buffered saline (PBS), and whole cell lysates were prepared. Twenty micrograms of proteins was resolved using 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes by electroblotting. The membranes were blocked with 5% non-fat milk powder (BioRad) and incubated overnight with a primary antibody at 4°C. The membranes were probed with secondary antibodies conjugated with horseradish peroxidase (HRP). Finally, the bands were visualized by adding Super Signal West Dura Extended Duration Substrate (Thermo Scientific) according to the manufacturer’s instructions. The images obtained were used for quantification with the ImageJ software, as described previously [52 (link)]. Mouse monoclonal Runx2 (1:1,000) antibodies, Cdk2 (1:1,000) antibodies, and secondary antibodies conjugated with HRP were obtained from Santa Cruz Biotechnology. Cdk2 antibodies served as internal loading controls.

Gene expression was analyzed using western blot analysis. Cell pellets were lysed on ice in RIPA buffer (Pierce, Rockford, IL) supplemented with Complete Mini EDTA-free Protease Inhibitor Cocktail (Roche, Indianapolis, IN) and Phosphatase Inhibitor Cocktail (Sigma, St. Louis, MO). Protein concentrations were determined by Bradford assay (Bio-Rad, Hercules, CA). Protein (40 μg) was diluted 1:5 in 5X protein loading buffer (Fermentas, Glen Burnie, MD), boiled at 80 °C for 5 min, electrophoresed on a 4–20% Tris-Glycine gel, and transferred using a Trans-Blot Turbo Transfer System (Bio-Rad, Hercules, CA). Membranes were blocked in 5% Non-fat milk powder (BioRad), incubated with primary antibody overnight at 4 °C, incubated with HRP-coupled secondary antibody 1 h at room temperature, developed with Visualizer Western Blot Detection Kit (Millipore, Billerica, MA), and visualized on a LAS-4000 imager (Fujifilm, Edison, NJ). The following antibodies were used at 1: 1000 dilutions: human anti-CD44 (#3570S, Cell Signaling Technology) and mouse anti-actin (MAB 1501R, Millipore). Secondary antibody, anti-mouse-HRP (Santa Cruz Biotechnology, Santa Cruz, CA) was used at 1:10,000 dilutions.