Ecl plus system

Proteins were separated via SDS-PAGE and transferred onto a PVDF membrane (Sigma, St. Louis, MO, USA, cat. no. IPVH00010). Subsequently, the membrane was blocked with 5% skimmed milk (Fdbio Science, Hangzhou, China, cat. no. FD0080) in TBST buffer at room temperature for 1 h. Following the blocking step, the membrane was incubated with primary antibodies overnight at 4 °C on a shaker, followed by three washes with TBST. Next, the membrane was incubated with secondary antibodies at room temperature for 1 h on a shaker, and then washed three times with TBST. Finally, protein bands were visualized using an enhanced chemiluminescence (ECL) Plus system (Vazyme, Nanjing, China, cat. no. L/N 7E530C1) and scanned with a ChemiScope series Imaging System (Clinx Science Instruments Co., Ltd., Shanghai, China, cat. no. 6300).
Primary antibody: OCT4 (SantaCruz, SantaCruz, California, USA, cat. no. SC-5297, 1:1000), SOX2 (Abcam, Cambridge, UK, cat. no. Ab97959, 1:500), NANOG (Abcam, Cambridge, UK, cat. no. Ab80892, 1:1000), STAT3 (SantaCruz, SantaCruz, California, USA, cat. no. SC-8019, 1:500), p-STAT3 Tyr705 (CST, Leiden, Netherlands, cat. no. 9131s, 1:1000), MPP8 (proteintech, Wuhan, China, cat. no. I6796-I-AP, 1:500).