Grp75

Cells were lysed in 62.5 mmol/L Tris (pH 6.8)-2% sodium dodecyl sulphate (SDS) mixed with the protease inhibitor cocktail (Sigma) and briefly sonicated before determining the protein concentration using the BCA reagent (Pierce, Rockford, IL, USA). A 50 µg of protein was resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride membrane filter (Bio-Rad, Hercules, CA, USA). Membrane filters were blocked in 0.1 M Tris (pH 7.5)-0.9% NaCl-0.05% Tween 20 with 5% nonfat dry milk, and incubated with appropriate antibodies. Antibodies were diluted as follows: glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 1:5,000; poly(ADP-ribose) polymerase (PARP), 1:1,000; cleaved lamin A, 1:2,000; β-actin, 1:10,000; cytochrome c oxidase (COX IV), 1:2,000 (Cell Signaling, Danvers, MA, USA); RET, 1:1,000; TP53, 1:1,000; GRP75, 1:2,000 (Santa Cruz Biotechnology, Santa Cruz, CA, USA); E2 promoter binding factor 1 (E2F-1), 1:1,000 (NeoMarkers, Fremont, CA, USA); Ki-67, 1:1,000 (Dako, Glostrup, Denmark); peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α), 1:2,000 (Abcam, Cambridge, UK). The Supersignal West Femto and Pico chemiluminescence kits (Pierce) were used for visualization of the signal. Images of immunoblots were taken and processed using ChemiDoc XRS+ and Image Lab 3.0 (Bio-Rad).