Anti β tubulin e7

Cell lysis was carried out identically for SH-SY5Y cells, HEK293 cells and human fibroblasts as well as MEFs. Cells were washed with phosphate-buffered saline (PBS) and detached with Trypsin at 37°C for 3 min. After 5 min of centrifugation at 14 000 r.p.m., cells were lysed in buffer containing 0.1% Triton X-100 in PBS and 1 × protease inhibitor cocktail (Roche, Penzberg, Germany). Proteins were detected by using antibodies against mortalin (anti-GRP75, Santa Cruz Biotechnology, Dallas, TX, USA), β-Actin (Sigma-Aldrich, Munich, Germany), GAPDH (Life Technologies, Carlsbad, CA, USA), Tom20 (Santa Cruz Biotechnology), Hsp60 (from A Azem, Tel-Aviv University, Ramat Aviv, Israel), activated caspase 3 (Cell Signaling Technology, Danvers, MA, USA), activated caspase 9 (Cell Signaling Technology), Flag (Sigma-Aldrich), α-tubulin (Sigma-Aldrich), β-Tubulin (anti-β-Tubulin, (E7), Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA, USA), GAPDH (anti-GAPDH (G9545), Sigma-Aldrich) and rodent OXPHOS (no. MS604 Mitoscience (Abcam), Cambridgeshire, UK). Secondary antibodies were purchased from GE Healthcare (Buckinghamshire, UK).
Densitometry from western blot was performed using the ImageJ 1.41o software (Wayne Rasband; National Institutes of Health, Bethesda, MD, USA).

All protein samples were prepared from SK-N-MC cells by using SDS lysis buffer (100 mM Tris–HCl, pH 6.8, 2% SDS, 40% glycerol, 5% β-mercaptoethanol and 0.1% bromophenol blue). Samples were pre-heated at 99°C for 10 min before loaded onto a 12% SDS-PAGE gel. Primary antibodies used in this study were: anti-nucleolin 3G4B2 (Sigma-Aldrich; 1:1000) for endogenous NCL, anti-GFP (JL-8, Clontech; 1:2000) for EGFP-NCL, anti-Akt1 (B-1, Santa Cruz; 1:1000) for Akt1, anti-p53 (1C12, Cell Signaling Technology; 1:2000), anti-β-tubulin (E7, Developmental Studies Hybridoma Bank; 1:5000). HRP conjugated Goat anti-Rabbit IgG (H + L) (11-035-045) and HRP conjugated Goat anti-Mouse IgG (H + L) (115-035-062) secondary antibodies (Jackson ImmunoResearch) were used at 1:5000. The chemiluminescence signal was developed using Immobilon Forte Western HRP substrate (Merck Millipore), and the images were captured and processed using ChemiDoc™ Touch Imaging System (Bio-Rad).