Fibronectin Antibody (617): sc-81769

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	-Mouse (1:2000) -Use clean forceps to gently handle the blot from the corner without creasing the membrane. Do not write on the blot with pen or marker, as the ink can fluoresce and cause background.

Protocol tips
-Mouse (1:2000) -Use clean forceps to gently handle the blot from the corner without creasing the membrane. Do not write on the blot with pen or marker, as the ink can fluoresce and cause background.

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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