Horseradish peroxidase conjugated anti rabbit igg

Fifty micrograms of proteins from each experimental group were applied to Bolt 4–12% Bis-Tris Plus gels (Invitrogen, Karlsruhe, Germany) and electrophoresed for 2 h 30 min at 80 V. Proteins were transferred onto a PVDF membrane in blotting buffer for 1 h at 100 V and blocked with 5% skim milk (Difco, Detroit, MI, USA) or 5% BSA (Gibco, Grand Island, NY, USA) in TBST for 1 h at room temperature. The blotted membrane was then incubated overnight at 4 °C with the different primary antibodies. Antibodies against GMPPA (1:4,000) and RRM2 (1:5,000) were purchased from Young in Frontier (Seoul, Korea), MAVS (1:5,000) was from Bethyl Lab (Montgomery, TX, USA), SOD1 (1:1000) and IPO4 (1:1000) were from Invitrogen (San Diego, CA, USA) and β-actin (1:10,000) was from Cell Signaling Technology (Beverley, MA, USA). Blots were then incubated with horseradish-peroxidase conjugated anti-rabbit IgG (GeneTex, Irvine, CA, USA, diluted 1:7,000 for GMPPA, 1:5,000 for MAVS and IPO4, Jackson ImmunoResearch, West Grove, PA, USA, diluted 1:10,000 for SOD1) and anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA, USA, diluted 1:11,000 for RRM2) for 1 h at room temperature. Detection was performed using an ECL system (Amersham Pharmacia Biotech, Piscataway, NJ, USA).

The hDF were loaded into hydrogels and collected based on similar methods to those described above. The collected cells were then washed with cold phosphate-buffered solution and treated with radioimmunoprecipitation assay buffer (Abcam, Cambridge, UK) and cold centrifuged a 14,000 rpm for 20 min. A Bradford protein assay from Bio-Rad, Richmond, CA, USA was used to evaluate for the levels of the different proteins. According to the manufacturer’s instructions, SDS-PAGE was used to separate the proteins, which were subsequently transferred onto polyvinylidene difluoride membranes. Target primary antibodies (β-actin, Abcam; MMP2, Millipore, Billerica, MA, USA; MMP9, Abcam; Decorin, Abcam) were placed onto the membranes and incubated overnight. Then, the membranes were washed and incubated with either horseradish peroxidase-conjugated anti-rabbit IgG (1:2,000 dilution; Genetex, Hsinchu, Taiwan) or horseradish peroxidase-conjugated anti-mouse IgG (1:2,000 dilution; Genetex) for 1 h at room temperature. The Fusion-Solo chemiluminescence system (Vilber, Paris, France) and ECL Western blotting Detection Reagents (Thermo Fisher Scientific, Waltham, MA, USA) were then used to detect the signals emitted from the samples.

The hDF samples were seeded on the Si-GelMa hydrogels for different time points and washed three times with PBS. Then, the cells were lysed with an NP40 buffer (Invitrogen) to assess the protein concentrations using a bicinchoninic acid protein assay kit (Invitrogen). SDS-PAGE was used to separate the cell lysates (40 μg protein) according to the manufacturer’s instructions. They were subsequently transferred onto polyvinylidene difluoride membranes. Target primary antibodies (phospo-extracellular signal-regulated kinases 1/2 (pERK1/2), extracellular signal-regulated kinases 1/2 (ERK1/2), phospo-p38, p38, β-actin, Col I, Ki67, MMP9 and Decorin (Abcam) were placed onto the membranes and incubated overnight. Then, the membranes were washed and incubated with either horseradish peroxidase-conjugated anti-rabbit IgG (1:2000 dilution; Genetex, Hsinchu, Taiwan) or horseradish peroxidase-conjugated anti-mouse IgG (1:2000 dilution; Genetex) for 1 h at room temperature. Then, the Fusion-Solo chemiluminescence system (Vilber, Paris, France) and ECL detection reagents (Thermo Fisher, Waltham, MA, USA) were used to detect the signals emitted from the samples.