Anti phospho erk tyr204

Western blot analysis was performed as described previously [21 (link)]. The primary antibodies used for Western blot analyses included anti-phospho-Src (Tyr418) (Invitrogen, Carlsbad, CA, USA), anti-phospho-FAK (Tyr576) (Invitrogen), anti-FAK (Invitrogen), anti-GAPDH (Invitrogen), anti-phospho-EGFR (Tyr1068) (Cell Signaling, Beverly, MA, USA), anti-phospho-STAT3 (Tyr705) (Cell Signaling), anti-phospho-PI3K (Tyr458) (Cell Signaling), anti-AKT (Cell Signaling), anti-phospho-SAPK/JNK (Thr183/Tyr185) (Cell Signaling), anti-SAPK/JNK (Cell Signaling), anti-phospho-Paxillin (Tyr118) (Cell Signaling), anti phosphor-p130Cas (Tyr410) (Cell Signaling), anti-EGFR (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-STAT3 (Santa Cruz Biotechnology), anti-PI3K (Santa Cruz Biotechnology), anti-phospho-MEK1/2 (Ser218/Ser222) (Santa Cruz Biotechnology), anti-MEK (Santa Cruz Biotechnology), anti-phospho-ERK (Tyr204) (Santa Cruz Biotechnology), anti-ERK2 (Santa Cruz Biotechnology), anti-Paxillin (Santa Cruz Biotechnology), anti-p130 Cas (Santa Cruz Biotechnology), anti-phospho-AKT (Ser473) (Millipore, Billerica, MA, USA)

Cells were seeded into 100 mm Petri dishes at 1 × 10⁶ cells/plate. The samples were treated with 3a at 5 and 10 μM for 24 or 48 h. Cellular homogenate was obtained using RIPA lysis buffer with protease and phosphatase inhibitors (Sigma, #P8340). After centrifugation (10,000× g for 10 min at 4 °C), supernatants were collected, and quantification of total proteins was determined using BCA kit (Pierce Biotechnology Inc., Waltham, MA, USA). 50 μg of total protein was separated by SDS–PAGE (12%) and transferred (100 V, 250 mA for 2 h) onto a PVDF membrane (Amersham Bioscience, Amersham, UK). After membrane blocking (5% non-fat milk in Tris-buffered saline + 0.1% Tween20) (1 h at 4 °C), it was probed with primary antibodies: anti-phospho-ERK(Tyr 204) (Santa-Cruz, sc-7383, 1:200), anti-ERK 1/2 (Cell signaling, #4696, 1:1000), anti-Cyclin B1 (Santa Cruz, sc-245, 1:200), anti-AKT(Ser 473) (Cell signaling, #4060), and α-tubulin (Sigma–1:1000) overnight at 4 °C. After washing, the membrane was incubated with an appropriate secondary antibody—HRP conjugated for 2h at room temperature. Immunoreactive bands were revealed by ECL Western blotting Detection Kit (Amersham Bioscience), and quantified by ImageJ [73 (link)].