Bs 2067r

Total proteins were extracted from cells using RIPA buffer (Cell Signaling Technology) and quantified using the BCA Protein Quantification Kit (Abbkine, Wuhan, Hubei, USA). The same amount of protein was loaded and separated by SDS-polyacrylamide gel electrophoresis (SDS‒PAGE) and then transferred to a PVDF membrane. Subsequently, membranes were blocked with 5% nonfat milk for 1 h at RT and then incubated overnight at 4 °C with the primary antibody. The membranes were then washed with Tris-buffered saline containing 0.1% Tween 20 (TBST) and incubated with the corresponding secondary antibodies (1: 5000, BOSTER, Wuhan, Hubei, China) at RT for 1 h. Finally, the signals of the targeted proteins were detected by a chemiluminescence detection kit (Beyotime Biotechnology). The primary antibodies used in this study included NIPBL antibody (#ab245539, Abcam), RAD21 antibody (#4321, Cell Signaling Technology), lysine demethylase 6B (KDM6B) antibody (#ab38113, Abcam), EZH2 antibody (#21800-1-AP, Proteintech), PI3K antibody (#bs-2067R, Bioss, Beijing, China), phospho-PI3K (Tyr317) antibody (#bs-5570R, Bioss) and GAPDH (#KC-5G5, Aksomicks, Shanghai, China).

After 7 and 14 days of culture, the TSPCs in Groups N and G with (N+ and G+) or without (N- and G-) phosphoinositide 3-kinase (PI3K)/AKT pathway inhibitor (LY 294002; Bioss) were lysed in lysis buffer containing a mixture of proteinase inhibitors (Thermo Fisher Scientific). Equal amounts of protein samples (30 μg/lane) were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrotransferred to polyvinylidene difluoride membranes. Then, the membranes were incubated overnight at 4°C with the following primary antibodies: anti-TNC (1: 1000, ab108930; Abcam, Cambridge, UK), anti-TNMD (1: 1000, ab203676; Abcam), anti-AKT (1: 1000, 9272S; Cell Signaling Technology (CST), Danvers, MA, USA), anti-phospho-AKT (pAKT473, 1: 1000, 9271S; CST), anti-phospho-AKT (Thr308) (pAKT308, 1: 1000, 9275S; CST), anti-PIK3CA (PI3K, 1: 1000, bs-2067R; Bioss), anti-phospho-PI3KCA (p-PI3K, 1 : 1000, bs-5570R; Bioss), anti-integrin α2 (1 : 1000, bs-52613R; Bioss), anti-integrin β1 (1 : 1000, bs-0486R; Bioss), and anti-GAPDH (1 : 1000, ab8245, Abcam). GAPDH was used as a loading control. Then, the membranes were incubated with secondary antibody (10285-1-AP, 1 : 2000; Proteintech, Wuhan, China) for 2 h at room temperature. The protein bands were visualized by enhanced chemiluminescence (GE Healthcare, Wuxi, China), and the results were analyzed with ImageJ software.