Fuji rx film

Cell pellets from synchronized HeLa CCL2 cells and samples from pulldown experiments were resuspended in SDS-sample buffer and briefly denatured at 95°C. Protein was resolved by SDS-PAGE and transferred to nitrocellulose blotting membranes (GE Healthcare). Membranes were blocked over night with 5% skim milk powder in PBS-T (PBS containing 0.1% Tween 20). Subsequently, membranes were incubated at RT for 1 h with indicated antibodies diluted in 5% milk-PBS-T. Primary rabbit polyclonal antibodies directed against NUP188, NUP93, NUP53 and NUP54 have been described (Linder et al., 2017 (link)). Antibodies directed against actin (Sigma, cat no. A1978), HA (Roche), pH3 (Cell Signaling, cat no. 9701S) and NUP62 (Abcam, cat no. ab188413) are commercially available. After three washing steps with TBS-T secondary antibody solutions were applied in 5% milk-PBS-T and membranes kept shaking for 1 h at RT. Subsequent washing was followed by detection. HRP-conjugated secondary antibodies used to detect primary antibodies included goat anti–rabbit IgG and goat anti-mouse IgG (Sigma-Aldrich). Chemiluminescence was initiated using ECL detection reagent (GE Healthcare) and the signal was detected using Fuji RX film (Fujifilm)

Cell lysates and immunoblotting were performed as previously described [32 (link)]. BMMCs were harvested, lysed in lysis buffer (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, and proteinase inhibitor cocktail (Roche, Basel, Swizerland) for 30 min at 4 °C, and spun at 12,000× g at 4 °C for 30 min. The eluted and reduced samples were resolved by SDS-PAGE using a 5-20% gradient polyacrylamide gel (Wako Pure Chemical, Osaka Japan) and transferred to a PVDF membrane (Immobilon-P, Millipore, Billeria, MA, USA). For immunoblotting, the membranes were incubated with primary antibodies for 1 h at room temperature, and then incubated with HRP-conjugated anti-mouse (Thermo Fisher Scientific) or anti-rabbit (Thermo Fisher Scientific) antibody for 1 h at room temperature. After extensive washing of the membranes, immunoreactive proteins were visualized using the Western Lightning-ECL system (GE Healthcare Life Sciences, Buckinghamshire, England) according to the manufacturer’s recommendation. The PVDF membranes were exposed to Fuji RX film (Fujifilm). Densitometric analysis was performed using a LAS-4000 fluorescence image analyzer (Fujifilm, Tokyo, Japan).

DNA samples were screened for mutations of K-ras by PCR-single-strand conformation polymorphism (SSCP) analysis. The PCR primers were designed to amplify mutation hot spots, codons 12 and 13, of the K-ras [8 (link)]. PCR-SSCP analysis was performed as described previously [8 (link)]. Briefly, each 25-_μL reaction mixture contained 1 × AmpliTaq Gold Buffer (8.0 mmol/L Tris–HCl, pH 8.3; 40 mmol/L KCl; Perkin-Elmer, Branchburg, NJ, USA), 4 mmol/L MgCl₂, 0.3 mmol/L of each deoxynucleotide triphosphate, 100 pmol of each primer, 10–20 ng genomic DNA, 2.5 mCi [_α³²-P]dCTP (3000 Ci/mmol/L, 10 mCi/mL), and 1.25 U AmpliTaq Gold DNA polymerase (Perkin-Elmer). The reaction mixtures were heated to 95°C for 10 min, followed by 45 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 2 min, and strand elongation at 72°C for 2 min. After PCR, the samples were electrophoresed on 6% polyacrylamide gels (ratio of acrylamide:bis-acrylamide, 19:1) with 10% glycerol at 4°C. The gels were then subjected to autoradiography overnight at −80°C on Fuji RX film (Fuji Film, Minamiashigara, Japan).