Ab109290

Cells were lysed in RIPA lysis buffer (50 mM Tris–HCl, 150 mM NaCl, 1 mM EDTA-Na2, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS) supplemented with protease inhibitors and phosphatase inhibitor on ice for 15 min. The cell lysates were cleared by centrifugation at 12,000×g for 10 min, then heat-denatured with 5 ×  Laemmli buffer (G2013, Servicebio). The prepared samples with equal amount of total protein (20 μg) were subjected to SDS-PAGE and immunoblotting, as per the standard procedure. The following antibodies were used: anti-SOX1 (ab109290, Abcam), anti-β-catenin (ab32572, Abcam), anti-HES1 (#11988, Cell Signaling Technology [CST]), anti-PROX1 (11067-2-AP, Proteintech), anti-p38 (#8690, CST), anti-phospho-p38 (#9215, CST), anti-ERK (#4695, CST), anti-phospho-ERK (#4370, CST), anti-AKT (#4691, CST), anti-phospho-AKT (#4060, CST), anti-JNK (#9252, CST), anti-phospho-JNK (#700031, Invitrogen), anti-RAF (#9422, CST), anti-phospho-RAF (#9427, CST), anti-MEK (51080-1-AP, Proteintech), anti-phospho-MEK (#9154, CST), anti-α-Tubulin (11224-1-AP, Proteintech), anti-GAPDH (BM1623, Boster), anti-BCL2 (Proteintech, 12789-1-AP), anti-PCNA (BM0104, Boster). All antibodies were diluted by antibody dilutions (G2025, Servicebio) with a ratio of 1:1,000. All quantitative analyses were performed using the software image J.

RIPA (Beyotime, Shanghai, China) was applied to isolate total protein. BCA (Thermo Fisher Scientific) was used to quantify the total protein. SDS-PAGE (10%) was used to separate proteins (40 μg per lane), and then proteins were transferred onto PVDF membranes (Thermo Fisher Scientific). The membranes were incubated with primary antibodies against SOX1 (1:1000, #ab109290, ABCAm, Cambridge, UK) and β-actin (1:5000, #ab8226, ABCAm, Cambridge, UK) at 4 °C overnight after being blocked with 5% skimmed milk for 1 h. After that, the membranes were incubated with secondary antibodies HRP-conjugated (1:5000, #ab20272, ABCAm, Cambridge, UK) for 1 h at room temperature. Protein bands were visualized using the enhanced chemiluminescent (ECL) kit (Thermo Fisher Scientific). β-actin was used for normalization. Image-Pro Plus 6.0 was applied for densitometric analysis.

Doxycycline (HY-N0565B) and CFSE (HY-D0938) were purchased from Medchem Express (MCE). Bovine serum albumin (BSA, FC0077) and propidium iodide (PI, 219545810) were purchased from MP Biomedicals. RNase A (10406ES03) was purchased from Yeasen Biotechnology. 5-Bromo-2′-deoxyuridine (BrdU, B5002), Hoechst 33342 (B2261), Triton X-100 (T8787), Pyronin Y (213519), and 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI, D9542) were purchased from Sigma-Aldrich. Paclitaxel was purchased from Beijing SL Pharmaceutical Co., Ltd. (China). Cisplatin was purchased from Jiangsu Hansoh Pharmaceutical Co., Ltd. (China).
Antibodies against SOX1 (ab109290) and RPS3 (ab128995) were purchased from Abcam. Antibody against RPL7A (DF9137) was purchased from Affinity Biosciences. Antibodies against β-actin (60008-1-Ig), Ki-67 (27309-1-AP), and c-MYC (10828-1-AP) were purchased from Proteintech. Antibody against p27 Kip1 (sc-528) was purchased from Santa Cruz Biotechnology. Alexa Fluor 488 cross-adsorbed goat anti-rabbit IgG secondary antibody (A11008) was purchased from Invitrogen.