Goat anti rabbit horseradish peroxidase hrp secondary antibody

Day 9 cells were lysed in RIPA buffer containing protease and phosphatase inhibitors (Thermo Fisher) and then centrifuged at 12,000g for 20 min. Protein concentration of the supernatants was determined using a BCA protein assay kit (Thermo Fisher), with bovine serum albumin (BSA) as a standard. Proteins (10 μg) were electrophoresed onto 5%–20% TBX gels (Bio-Rad, Hercules, CA). Immunoblotting was performed according to standard methods, using primary antibodies for Sox2 (1:1000, Abcam, #ab97959), Smad2/3 (1:1000; Cell Signaling Technology, Danvers, MA #8685), or phospho-Smad2/3 (1:1000; Cell Signaling Technology, #8828) and goat anti-rabbit horseradish peroxidase (HRP) secondary antibody (1:2000–1:5000, Santa Cruz Biotechnology, #sc-2004). Protein expression was examined by chemiluminescence (Luminol, Santa Cruz Biotechnology) on a Bio-Rad ChemiDoc imaging system. Densitometry values were determined using Bio-Rad Image Lab software and normalized to Gapdh (1:1000, GeneTex, Irvine, CA, #GTX627408) as a loading control using goat anti-mouse-HRP secondary antibody (1:2000–1:5000, Invitrogen, #62–6520). Densitometry values were compared with the average control value to calculate individual fold changes. Statistical differences were determined using Student’s t-test (p ≤ 0.05) (n = 3).