Anti tgf β receptor 1

Western blot analysis was performed as previously described by us [23 (link)]. The following antibodies were used in the analysis: anti-EVI5 (Millipore, Billerica, MA, USA); anti-Emi1 and anti-TGF-β receptor II (Santa Cruz, CA, USA); anti-CyclinA2 (Proteintech, IL, USA); anti-pAkt (Ser473), anti-Akt, anti-Erk1/2, anti-pErk (Thr202/Tyr204), anti-CyclinD1, anti-MMP2, anti-p-Smad3, anti-Snail and anti-β-actin (Cell Signaling Technology, Danvers, MA, USA); anti-TGF-β receptor I (Abcam, London, UK); anti-N-cadherin and anti-Vimentin (BD Biosciences, USA); Anti-mouse and anti-rabbit secondary antibodies (Cell Signaling Technology, Danvers, MA, USA).

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).