Actn05 c4

Cells were lysed in Triton-X lysis buffer containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5% Triton-X-100, 1 mM phenylmethylsulfonyl fluoride, 1 mM DTT, and 1 × Complete protease inhibitor cocktail (Roche), after which cellular debris was removed by centrifugation. Samples were resuspended in 4× Laemmli sample buffer and denatured at 95 °C. Proteins were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gel and immobilized onto Nitrocellulose-membranes (Thermo Fisher Scientific). Primary antibodies against PDK4 (PA5-13776, Thermo Fischer Scientific), and actin (ACTN05 C4, Abcam, Cambridge, UK), were used together with anti-mouse or anti-rabbit HRP-conjugated antibodies produced in goats (Invitrogen, Thermo Fisher Scientific). Chemiluminescence reactions were generated using Clarity Western ECL Substrate reagent (Bio-Rad Laboratories) and measured using ChemiDoc MP Imaging system (Bio-Rad Laboratories). Quantitation of signals was performed using Image Lab software (Version 6.0.0 build 25, Bio-Rad Laboratories).

Cells were treated with Oxaliplatin (NSC 266046, Selleck Chemistry, Houston, TX, USA), Cisplatin (Calbiochem, Sigma Aldrich), Carboplatin (Tocris) and Actinomycin D (Sigma Aldrich) in different dosages for 24 h. The primary antibodies used were anti-NPM mouse monoclonal antibody (E-3, Santa Cruz Biotechnology, CA, USA), anti-Coilin rabbit monoclonal antibody (D2L3J, Cell Signaling Technology, Leiden, The Netherlands), anti-SMN1 mouse monoclonal antibody (2F1, Cell Signaling Technology), anti-Fibrillarin rabbit monoclonal antibody (C13C3, Cell Signaling Technology), anti-FUS mouse monoclonal antibody (4H11, Santa Cruz Biotechnology), anti-TARDBP (TDP43) mouse monoclonal antibody (2E2-D3, Abnova), anti-actin mouse monoclonal (ACTN05 C4, Abcam, Cambridge, UK), anti-tubulin mouse monoclonal antibody (TU-02, Santa Cruz Biotechnology) and anti-lamin rabbit polyclonal antibody (B1, ab16048, Abcam).