Publication protocol
For total protein extraction, cells were lysed with 100 µl radioimmunoprecipitation assay lysis buffer with protease inhibitor cocktail (Invitrogen; Thermo Fisher Scientific, Inc.). The cell lysates were collected following centrifugation (6,000 × g for 10 min) and mixed with 5X sodium dodecyl sulfate (SDS) sample buffer (Takara Biotechnology, Inc., Dalian, China), and a bicinchoninic acid assay was used to measure the protein levels. The samples (40 µg) were loaded and separated on 10% SDS-PAGE (Takara Biotechnology, Inc.), and then transferred to polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA). The membranes were blocked in 5% non-fat milk and incubated with the following primary antibodies at 4°C overnight: anti-TAK1 (1:2,000; sc-7967), anti-α-smooth muscle actin (SMA; 1:1,500; sc-53015), anti-fibronectin (1:2,000; sc-81769), anti-NF-κB p65 (1:2,000; sc-8008), anti-IKKα (1:2,000; sc-7606), anti-p-Smad2 (1:2,000; sc-101801), anti-p-Smad3 (1:2,000; sc-130218) and GAPDH (1:1,500; sc-166574; all from Santa Cruz Biotechnology, Inc., Dallas, TX, USA). After washing with phosphate-buffered saline with Tween-20, the membranes were incubated with goat anti-mouse IgG horseradish peroxidase-conjugated secondary antibodies (1:3,000; sc-395760; Santa Cruz Biotechnology, Inc.) and goat anti-rabbit HRP-conjugated secondary antibodies (1:2,000; sc-2030; Santa Cruz Biotechnology, Inc.) for 1 h at room temperature. The bands on the membranes were visualized using chemiluminescence detection reagents (Roche Diagnostics GmbH, Mannheim, Germany). Densitometic analysis was conducted using Image J version 1.41 (National Institutes of Health, Bethesda, MD, USA). GAPDH was used as a control.
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