To detect the MAPK phosphorylation signalling pathways, the Meso Scale Discovery (MSD) MULTI-SPOT (MSD, Rockville, MD) protein detection assay using MESO QuickPlex SQ 120 (MSD).
Caco-2 cells grown to confluence on 6-well ThinCert inserts were treated apically with RGal (1000 μg ml À1 ) for 1, 15, 30 and 60 min or LPS (10 ng ml À1 ) for 30 min. After treatment, cells were washed with cold DPBS and incubated with lysis buffer (20 mM Tris.HCl pH 7.8, 100 mM NaCl, 0.1% SDS, 0.5% Triton-X, 1 mM EDTA, 50 mM NaF, 2 mM Na 3 VO 4, 1x protease inhibitor cocktail) for 30 min. The lysates were centrifuged at 20,000 Â g, 4 C by 10 min and the protein concentration quantified in supernatants with the colorimetric DC™ Protein Assay Kit (Bio-Rad, Hercules, CA, USA).
Ten micrograms of protein were added to the MSD MULTI-SPOT ® 96-well 4-spot plate and phospho-ERK1/2, total-ERK1/2, phospho-p38, total-p38, phospho-JNK, and total-JNK were determined using the Human MAP Kinase Panel (Phosphoprotein and Total Protein) Assay Kit (MSD) according to the manufacturer's protocol.
Caco-2 cells grown to confluence on 6-well ThinCert inserts were treated apically with RGal (1000 μg ml À1 ) for 1, 15, 30 and 60 min or LPS (10 ng ml À1 ) for 30 min. After treatment, cells were washed with cold DPBS and incubated with lysis buffer (20 mM Tris.HCl pH 7.8, 100 mM NaCl, 0.1% SDS, 0.5% Triton-X, 1 mM EDTA, 50 mM NaF, 2 mM Na 3 VO 4, 1x protease inhibitor cocktail) for 30 min. The lysates were centrifuged at 20,000 Â g, 4 C by 10 min and the protein concentration quantified in supernatants with the colorimetric DC™ Protein Assay Kit (Bio-Rad, Hercules, CA, USA).
Ten micrograms of protein were added to the MSD MULTI-SPOT ® 96-well 4-spot plate and phospho-ERK1/2, total-ERK1/2, phospho-p38, total-p38, phospho-JNK, and total-JNK were determined using the Human MAP Kinase Panel (Phosphoprotein and Total Protein) Assay Kit (MSD) according to the manufacturer's protocol.