Expression of the phosphorylated MAPK p38 and JNK (Jun amino-terminal kinases) was assessed on differentiated Caco-2 cells and jejunal explants by immunoblotting as previously described49 (link)54 (link). Cells differentiated on 24-well inserts or explants were treated with 10 μM of diluent (DMSO) or toxins, DON, deepoxy-DON or 3-epi-DON for 1 hour. Proteins were extracted, quantified and a total of 15 μg of protein was separated by SDS-PAGE. The membranes were probed with rabbit antibodies (Cell Signaling Technology, Danvers, USA) specific for: phospho-SPAK/JNK or phospho-p38 diluted at 1:500 or GAPDH diluted at 1:1000. After washing, the membranes were incubated with 1:10,000 CFTM770 goat anti-rabbit IgG (Biotium, Hayward, USA) for the detection. Antibody detection was performed using an Odyssey Infrared Imaging Scanner (Li-Cor Science Tec, les Ulis, France) with the 770 nm channel. The expression of the proteins was estimated after normalization with GAPDH signal.
Cftm770 goat anti rabbit igg
Manufactured by Biotium
Sourced in United States
The CFTM770 goat anti-rabbit IgG is a fluorescently labeled secondary antibody used for the detection and visualization of rabbit primary antibodies in various immunoassays and imaging applications.
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3 protocols using cftm770 goat anti rabbit igg
1
Phosphorylation of MAPK p38 and JNK in Caco-2 Cells
2
Quantification of Inflammatory Markers and p38 MAPK Activation
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Concentrations of TNF-α and IL-8 were measured in cell culture supernatants by enzyme linked immuno-absorbent assays (ELISA). Specific kits for porcine TNF-α and IL-8 (R&D Systems, Minneapolis, MN, USA) were used according to the manufactured instructions as already described20 (link), 66 (link).
The expression of phosphorylated p38 MAPK in IPEC J2 cell lysates was analyzed by western blot as already described26 (link). Briefly, total proteins were separated on SDS-PAGE, transferred onto nitrocellulose membranes, incubated with Rabbit anti-phospho-p38 (Cell Signaling Technology, Danvers, MA) overnight and further incubated with CFTM770 goat anti-rabbit IgG (Biotium, Hayward, CA). Mouse anti-β-actin (Cell Signaling Technology) was used as control. Membranes were analyzed using an Odyssey Infrared Imaging System (LI-COR; ScienceTec, Les Ulis, France). The expression of phosphorylated p38 MAPK was estimated after normalization with β-actin.
The expression of phosphorylated p38 MAPK in IPEC J2 cell lysates was analyzed by western blot as already described26 (link). Briefly, total proteins were separated on SDS-PAGE, transferred onto nitrocellulose membranes, incubated with Rabbit anti-phospho-p38 (Cell Signaling Technology, Danvers, MA) overnight and further incubated with CFTM770 goat anti-rabbit IgG (Biotium, Hayward, CA). Mouse anti-β-actin (Cell Signaling Technology) was used as control. Membranes were analyzed using an Odyssey Infrared Imaging System (LI-COR; ScienceTec, Les Ulis, France). The expression of phosphorylated p38 MAPK was estimated after normalization with β-actin.
3
MAPK Signaling Pathway in Caco2 Cells
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Caco2 cells differentiated on 24-well format (same process that for TEER), were treated with 10 µM of DMSO for control, D3G or DON for 1 hour to analyze the expression of the total and phosphorylated MAPK P38 and JNK. Proteins were extracted from cells as previously described (Pinton et al. 2009 (link)) quantified and 15 µg of total proteins was separated by SDS-PAGE. The membranes were probed with rabbit antibodies (Cell Signaling Technology, Danvers, USA) specific for: SAPK/JNK and phospho-SPAK/JNK or p38 and phospho-p38 diluted at 1:500 or GAPDH diluted at 1:1000. After washing, the membranes were incubated with 1:10,000 CF TM 770 goat anti-rabbit IgG (Biotium, Hayward, USA) for the detection. Antibody detection was performed using an Odyssey Infrared Imaging Scanner (Li-Cor Science Tec, les Ulis, France) with the 770nm channel. The expression of the proteins was estimated after normalization with GAPDH signal. Three independent experiments were proceeding for each cell culture condition.
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