Anti sestrin2

Antibodies used for immunoblotting included anti-sestrin2 obtained from Proteintech Group, anti-phospho-p70 S6 Kinase, anti-phospho-S6 Ribosomal Protein, anti-S6 Ribosomal Protein, anti-E-cadherin, anti-N-cadherin, and anti-TSC2 from Cell Signaling Technology, anti-p70 S6 Kinase from Santa Cruz Biotechnology, anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Aviva Systems Biology, and anti-β-Actin from Developmental Studies Hybridoma Bank. Rapamycin was obtained from LC Laboratories. Torin 1 was purchased from Cayman Chemical. N-acetyl cysteine (NAC) was purchased from Sigma-Aldrich.

Cells were washed with PBS and lysed in RIPA buffer with Protease Inhibitor Cocktail (Bimake) on ice. Cell lysates were centrifuged (12,000 rpm) at 4 °C for 20 min and then quantified using the BCA Protein Assay Kit (Beyotime). The lysate was denatured with sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) sample loading buffer (Beyotime), followed by SDS–PAGE and electrotransfer to polyvinylidene difluoride membranes (Millipore, Darmstadt, Germany). The membranes were incubated overnight at 4 °C with anti-Sestrin2 (Proteintech Group 10795-1-AP 1:1000), anti-CD44 (Proteintech Group 15675-1-1AP 1:1000), anti-Sox2 (Proteintech Group 11064-1-AP 1:1000), anti-Oct4 (Proteintech Group 11263-1-AP 1:1000), anti-CXC chemokine receptor 4 (Cxcr4) (Proteintech Group 11073-2-AP 1:1000), anti-β-catenin (Proteintech Group 51067-2-AP 1:1000) and anti-β-actin (Proteintech Group 20536-1-AP 1:1000) at the appropriate dilution and then incubated with the secondary antibody at room temperature for 2 h. The bands were visualized using BeyoECL Plus (Beyotime).