Amersham hybond c extra

MM6 cells were seeded at a density of 2 × 10⁵ cells/ml and incubated for 24 hours without or with 50 nM calcitriol, 1 ng/ml TGFβ and with the indicated inhibitors. Whole cell lysates were then separated by SDS-PAGE and were transferred to nitrocellulose membranes (Amersham Hybond™-C Extra, GE Healthcare, Freiburg, Germany). After 1 hour blocking, the membranes were incubated with the appropriate primary antibody against 5-LO (BD Biosciences, Heidelberg, Germany: 610695) and β-actin (Santa Cruz: sc-1616) overnight at 4°C. The membranes were washed with PBS/0.1%-Tween-20 and were incubated with infrared-dye conjugated secondary antibodies (LI-COR Biosciences, Bad Homburg, Germany) for 45 minutes at room temperature. Detection of the protein signals was performed with the Odyssey^® Infrared Imaging System (LI-COR Biosciences).

The protocol was similar to that described by Oliveira et al. [47 (link)] with modifications. The protein content of the cell lysate was determined by a bicinchoninic acid (BCA) protein assay reagent kit (Thermo Fisher Scientific Inc., Waltham, MA, EUA) according to the manufacturer’s protocol. For the Western blot analysis, protein samples (20 μg of protein) were mixed with Laemmli buffer (Bio-Rad Laboratories, Hercules, CA, USA) and subjected to SDS-PAGE (10%). The proteins were transferred onto a nitrocellulose membrane (Amersham Hybond-C extra; GE Healthcare, Chicago, IL, USA) and stained with Ponceau S to assess the efficacy of the transfer. The membranes were incubated with primary antibody overnight with agitation at 4 °C and subsequently with secondary rabbit horseradish peroxidase antibodies (Cell Signaling Technology, Danvers, MA, EUA) for 1 h at room temperature. The following primary and secondary antibody combinations were used for the respective proteins: anti-Caspase 3, anti-JNK, anti-phospho-JNK, anti-JAK2, anti-STAT3, and anti-phospho-STAT3. These antibodies and HRP-conjugated anti-rabbit IgG were obtained from Cell Signaling Technology (Danvers, MA, USA), and anti-β-actin was purchased from Sigma-Aldrich (St. Louis, MO, USA).

The protein content of the cell lysate was determined by a bicinchoninic acid (BCA) protein assay reagent kit (Thermo Fisher Scientific, MA, USA) according to the manufacturer’s protocol. Before the Western blot analysis, protein samples (20 μg) were subjected to SDS-PAGE and stained with Laemmli buffer to ensure equal loading of the proteins. Proteins were transferred onto a nitrocellulose membrane (Amersham Hybond-C extra; GE Healthcare, Chicago, IL, USA). The membranes were incubated overnight at 4 °C with primary antibodies and subsequently with secondary rabbit horseradish peroxidase antibodies for 1 h at room temperature. The following primary and secondary antibody combinations were used for the proteins: caspase 3, caspase 9, Bax, Bcl-2 and HRP-conjugated anti-rabbit IgG were obtained from Cell Signaling Technology (Beverly, MA, USA) and β-actin was purchased from Merck/Sigma-Aldrich (Merck KGaA, Darmstadt, Germany).