Multiplexed bead immunoassays

Phosphorylated and total levels of glycogen synthase kinase-3b (GSK3b), insulin receptor substrate 1 (IRS1), Akt and c-Jun N-terminal kinase (JNK) and serum and hepatic IL-2, IL-4, IL-6 and IL-10 were determined by multiplexed bead immunoassays (Bio-Rad Laboratories, Madrid, Spain and Millipore; respectively), following the manufacturer's recommendations. A minimum of 50 beads per parameter were analyzed in the Bio-Plex suspension array system 200 (Bio-Rad). Raw data (median fluorescence intensity, MFI) were analyzed with the Bio-Plex Manager Software 4.1 (Bio-Rad Laboratories).

Phosphorylated and total protein levels of Akt, cAMP response element-binding protein (CREB), extracellular signal-regulated kinase (ERK)1/2; glycogen synthase kinase 3β (GSK3β), mTOR, phosphatase and tensin homolog on chromosome 10 (PTEN) and 70 kDa ribosomal protein S6 kinase 1 (p70S6K1) were measured in duplicate by multiplexed bead immunoassays (Bio-Rad Laboratories, Madrid, Spain and Merck Millipore, Darmstadt, Germany) as reported (Khan et al., 2004) (link). Briefly, magnetic beads conjugated to the appropriate antibodies and tissue lysates (50 µl each) were incubated for 18 hours at 4ºC. Afterwards, wells were washed using a magnetic separation block (Millipore) and antibody conjugated to biotin (25 µl) was added. After incubation for 30 min at room temperature, beads were incubated during 30 min with 50 µl streptavidin conjugated to phycoerythrin. A minimum of 50 beads per parameter were analyzed in the Bio-Plex suspension array system 200 (Bio-Rad). Raw data (median fluorescence intensity, MFI) were analyzed with the Bio-Plex Manager Software 4.1 (Bio-Rad Laboratories). Mean intra-and inter-assay coefficients of variation were 8.3% and 11.9%, respectively.