p27 Kip1 Monoclonal Antibody (DCS-72.F6)

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	-Mouse (1:500)

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-Mouse (1:500)

Publication protocol

Western blotting assays were carried out to evaluate the expression of cell cycle control proteins cyclin D1 and p27Kip1. Briefly, four days after seeding, the cultures were MF- or sham-exposed, following the standard protocol. At 2 h of continuous MF-or sham-exposure, the proteins were extracted in hypotonic lysis buffer (10 mM Tris-HCL (pH 7.6), 100 mM potassium chloride (KCL), 1 mM EDTA, 1mM Ditiotreitol, 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 µgr/mL Leupeptin, 5 µgr/mL Pepstatin A, 100 mM sodium fluoride (NaF), 20 mM β–glycerolphosphate, 20 mM sodium Molibdate, 0.5% Tritón X-100 and 0.1% SDS), and their concentrations were determined through Bradford’s assay [84]. The protein lysates (60 μg per lane) were resolved on 10% SDS/PAGE gels and transferred to nitrocellulose membranes (Hybond ECL, GE Healthcare, Little Chalfont, Buckinghamshire, UK). The membranes were blocked, and incubated overnight at 4 °C with mouse monoclonal antibodies against cyclin D1 (1:200 dilution; NCL-CYCLIN1-GM, Novocastra Laboratories, Newcastle upon Tyne, UK) or against p27Kip1 (1:500; AHZ0452, BioSource). β-Actin (1:5000; A5441, Sigma) was used as loading control. After washing, the membranes were incubated with horseradish peroxidase-labeled anti-mouse secondary antibody (GE Healthcare) for 1 h at room temperature. The bands were visualized by enhanced chemiluminescence (ECL) following the manufacturer’s instructions (GE Healthcare). The blots were analyzed by densitometric assay using PDI Quantity One-4.5.2 software (Bio-Rad, Hercules, CA, USA). Immunoblot assays were also conducted to evaluate the expression of phospho-p38 and phospho-JNK under MF- or sham-exposure, following the standard protocol. The cultures were MF- or sham-exposed for 15, 30 or 60 min intervals. Activation of p38 and JNK were detected with specific anti-phospho p38 (1:1000; 44–4846G, BioSource) and anti p-JNK (1:1000; 4668, Cell Signaling, Barcelona, Spain) antibodies. Also, for p-38, a horseradish peroxidase conjugated Anti-Rabbit IgG secondary antibody (1:5000; GE Healthcare), followed by ECL Advance Western Blotting Detection Kit (GE Healthcare) reaction was used. As for JNK, fluorescently labeled secondary antibody IRdye 800 CW, conjugated goat (polyclonal) anti-rabbit (1:10,000; LI-COR, Bioscience, LI-COR, Lincoln, USA) was used. The fluorescence signal was detected and quantified with Odyssey infrared imaging system (LI-COR). Equal loading of proteins was confirmed by β-actin (Sigma) immunoblot. For semiquantitative analysis of immunoblot bands, the optical density (OD) of each band was measured and quantified by densitometry through computer imaging (Quantity-One, Biorad, Munich, Germany).
At least four experimental replicates were conducted for each of the proteins studied. Three MF-exposed dishes vs. three sham-exposed dishes per experimental run and per exposure period were used.



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