Oct 4 ab19857

Cells were lysed using a Protein Extraction kit (P0027; Beyotime Institute of Biotechnology, Beijing, China). Whole cell protein concentrations were measured using BCA reagent (P0010S; Beyotime Institute of Biotechnology). Lysates (25 µg total protein) were separated by 12% SDS-PAGE and transferred to polyvinylidene difluoride membranes. The membranes were incubated in blocking buffer (3% bovine serum albumin; Sigma-Aldrich; Merck KGaA) at room temperature for 1 h. Subsequently, the membranes were incubated at 4°C overnight with the following primary antibodies: GAPDH (ab9485; 1:2,500), Oct-4 (ab19857; 1:1,000), Nanog (ab109250; 1:1,000) and Sox2 (ab97959; 1:1,000; all purchased from Abcam, Cambridge, MA, USA). Goat anti-rabbit secondary antibody conjugated with peroxidase (BS13278; 1:5,000; Bioworld Technology, Inc., St Louis Park, MN, USA) was used as the secondary detection antibody at 25°C for 1 h. The immunoblots were then detected using an enhanced chemiluminescence reagent (Pierce; Thermo Fisher Scientific, Inc.) on the ChemiDoc XRS+ system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Images were analyzed using Image Lab software 3.0 (Bio-Rad Laboratories, Inc.).

Antibodies specific for pYAP-S127 (13008), α-tubulin (2144s), CREB (9197) and YAP (14074, WB) were purchased from Cell Signaling Technology (MA); YAP (sc-101199, IF/ChIP/Co-IP) were purchased from Santa Cruz Biotechnology (TX); and EpCAM (ab71916), SOX9 (ab185230), LAPTM4B (ab82810, WB/IF/IHC), Nanog (ab14959), pYAP (ab76252) and OCT4 (ab19857) were from Abcam (MA).

Detection of protein expression by western blot was carried out according to the established protocols described previously 14 (link). Western blot was performed using the following antibodies: SOX2 (sc-20088) and β-actin (sc-47778) antibodies were purchased from Santa Cruz Biotechnology (Dallas, Texas, USA). OCT4 (ab19857), NANOG (ab80892) and PFKFB4 (ab137785) antibodies were purchased from Abcam (Cambridge, UK). CD44 (3570), Flag (8146s) and CREB (9197s) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).