Thyroid cancer cells were incubated with ice-cold cell lysis buffer, scraped and centrifuged, and the supernatant was stored at −80 °C. Twenty-five micrograms of total protein lysate were suspended in reduced SDS sample buffer and the lysates were subjected to SDS–PAGE (4–12%). The separated proteins were transferred to a nitrocellulose membrane (Invitrogen, Life Technologies Corp., Carlsbad, CA, USA) by electrophoretic blotting.
Membranes were incubated overnight with primary antibody against β-actin (Sigma–Aldrich), p-AKT1/2/3 (Ser473), total AKT, p-ERK1/2, total ERK, cleaved caspase 3, phospho-p70S6K (Thr389), phospho-pS6 (Ser235/236), phospho-AMPK (Thr172), total AMPK, cytochrome c oxidase subunit 4 (COX4) (Cell Signaling Technology, Danvers, MA, USA), γH2AX, cyclin D1 and β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA).
Detection of proteins was performed using the Li-Cor Odyssey imaging system (LI-COR Biosciences, Lincoln, NE, USA). Image Studio Lite version 3.1 (Licor) was used for band densitometry measurement and quantification of protein levels.
Membranes were incubated overnight with primary antibody against β-actin (Sigma–Aldrich), p-AKT1/2/3 (Ser473), total AKT, p-ERK1/2, total ERK, cleaved caspase 3, phospho-p70S6K (Thr389), phospho-pS6 (Ser235/236), phospho-AMPK (Thr172), total AMPK, cytochrome c oxidase subunit 4 (COX4) (Cell Signaling Technology, Danvers, MA, USA), γH2AX, cyclin D1 and β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA).
Detection of proteins was performed using the Li-Cor Odyssey imaging system (LI-COR Biosciences, Lincoln, NE, USA). Image Studio Lite version 3.1 (Licor) was used for band densitometry measurement and quantification of protein levels.