Goat antimouse igg hrp conjugated secondary antibody

For Western blot analysis, 20 μg of total proteins extracted from HEKs were separated on 4%–20% Tris-Glycine gel (Invitrogen, Carlsbad, CA, USA) and transferred to the nitrocellulose membrane (Invitrogen). Membranes were blocked for 1 hour at room temperature in 5% non-fat milk. Primary antibodies were diluted with their respective blocking buffers and incubated overnight at 4°C. Antibodies against FLG (1:200 dilution, Cat# sc-66192, Santa Cruz Biotechnology Inc., Dallas, TX, USA), and β-actin (1:5,000 dilution, Cat# A-5441, Sigma-Aldrich) were used as primary antibodies. Washing was performed with TBS 0.1% Tween 20 (TBS-T) before addition of goat anti-mouse IgG HRP-conjugated secondary antibody (1:5,000 diluted, Cat# ab205719, Abcam) for 1 hour at room temperature. After incubating secondary antibody and washing with TBS-T, chemiluminescence detection was performed using a chemiluminescence kit (Amersham ECL Western Blotting Kit, GE Healthcare) and developed on Automatic X-RAY Film Processor JP-33 (JPI Healthcare, Korea). The densitometry of the detected protein bands was calculated using ImageJ software, version 1.52 (National Institutes of Health, Bethesda, MD, USA).