Anti foxl2

After treatment, the cells were harvested, lysed and their proteins subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) for immunoblotting with the respective antibodies. The protein bands were detected using an Amersham Imager 600 (GE Healthcare Life Sciences, Amersham, Buckinghamshire, UK). The following antibodies were used in this study: anti-p53 (sc-126, Santa Cruz, CA, USA), anti-FOXL2 [generated in our laboratory as previously described (29 (link))], anti-GAPDH (sc-47724, Santa Cruz), anti-Myc (#2276S, Cell Signaling Technology, Danvers, MA, USA), anti-FLAG (F1804, Sigma-Aldrich), anti-p21 (sc-397, Santa Cruz), anti-PARP1 (sc-74469, Santa Cruz), anti-BAX (sc-493, Santa Cruz) and anti-Caspase 3 (9662, Cell Signaling Technology).

We isolated protein lysates from NSCLC cells via RIPA buffer (Cell Signaling Technology). The extract was centrifuged at 12,000 rpm at 4°C for 15 min after 10 min of vibration and then stored at −80°C. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis was used to resolve the protein lysates. Subsequently, the samples were transferred to a nitrocellulose membrane (Millipore). After 2 h of transfer, the membranes were immersed in 5% BSA in Tris buffer saline with Tween 20 for 1 h and then immunoblotted with specific primary antibodies for more than 12 h at 4°C. The bands were immunoblotted with matched secondary antibodies for 2 h at 15–25°C on the second day. An enhanced chemiluminescence kit (Thermo Fisher) was used to visualize the bands. β‐Actin protein levels were used to normalize sample loading. The antibodies used in this research were as follows: anti‐FOXL2, anti‐TGF‐β receptor II, and anti‐MMP9 (Santa Cruz); anti‐TGF‐β receptor I (Abcam); anti‐p‐Smad3 (Ser423/425, C25A9), anti‐pAkt (Ser473, D9E), anti‐pErk (Thr202/Tyr204, D13.14.4 E), anti‐Smad3, anti‐Akt, anti‐Erk, anti‐CyclinD1, anti‐MMP2, anti‐Snail, and anti‐β‐actin (Cell Signaling Technology); anti‐N‐cadherin, anti‐E‐cadherin and anti‐Vimentin (BD Biosciences); and anti‐mouse and anti‐rabbit secondary antibodies (Cell Signaling Technology).