Mouse anti lamin b

Human anti-CD44-PE Antibody, human anti-CD133-PE Antibody, and FcR Blocking Reagent were purchased from MACS Miltenyi Biotech (Bergisch Gladbach, Germany). Mouse anti-CKII (#sc-373894) and mouse anti-LAMIN B (#sc-365214) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA), rabbit anti-p14ARF (#SAB4500073) was from Sigma-Aldrich ApS (Søborg, Denmark) and human anti-TOP1 (#TG2012) was from TopoGEN (Buena Vista, CO, USA). Horse radish peroxidase, HRP conjugated goat anti-mouse, goat anti-human, and goat anti-rabbit were all purchased from DAKO (ThermoFisher Scientific, Roskilde, Denmark). Phi29 DNA polymerase and PrestoBlue reagent were purchased from Thermo Fisher Scientific (Roskilde, Denmark). All chemicals, cells media, and media components were purchased from Sigma-Aldrich ApS (Søborg, Denmark). α³²P-dATP was purchased from PerkinElmer (Skovlunde, Denmark).

Nuclear and cytoplasmic extract were prepared using the NE-PER^® Nuclear and Cytoplasmic Extraction reagents (Thermo Scientific) as per manufacturer’s instruction. Protein concentration was quantified using the BCA protein assay kit (Pierce). Equal amounts of protein were loaded in each lane and separated on a 10%, 12% SDS-PAGE, then transferred onto a nitrocellulose membrane. Membranes were probed with the following primary antibodies: mouse anti-eIF6 monoclonal, rabbit anti-Tubulin (1:1000 Cell Signaling), mouse anti-LaminB (1:1000 Santa Cruz), mouse β-actin.

Protein measurements were carried out using the Pierce BCA Protein Assay Kit (Thermo Scientific) in accordance with the manufacturer’s instructions. Samples were mixed with Laemmli’s loading buffer, boiled for 5 min, and subjected to SDS-PAGE (12%) followed by blotting onto nitrocellulose membranes for 30 min at 25 V using the Mini Trans-Blot Cell (Bio-Rad). Membranes were blocked for 1 hr with 5% non-fat milk in TBS at room temperature and subsequently probed overnight at 4°C with the primary antibody (1:1000). The following primary antibodies were used: rabbit anti-vinculin, rabbit anti-hexokinase-II, rabbit anti-Na⁺/K⁺-ATPase, rabbit anti-cleaved caspase-3 (all from Cell Signaling), mouse anti-caspase-2, mouse anti-caspase-3, mouse anti-PARP1, mouse anti-cytochrome c (all from BD Transduction Lab), mouse anti-lamin B, rabbit anti-Endonuclease G, mouse anti-AIF (all from Santa Cruz Biotech), mouse anti-caspase-8 (Enzo Life Science), rabbit anti-GAPDH (Trevigen), and rabbit anti-ERp29 (kindly provided by Dr. S. Mkrtchian, Karolinska Institutet). After four times washes in TBST (0.05% Tween-20 in TBS), membranes were incubated with appropriate horseradish peroxidase-conjugated secondary antibodies purchased from Cell Signaling (1:4000) for 1 hr at room temperature. Blots were developed using ECL (Amersham Biosciences) and documented using Chemi-Doc (Bio-Rad).