Sandwich elisa

Phospho-Smad 2/3 was determined using the PathScan® Phospho-Smad 2 (Ser465/467)/Smad 3 (Ser423/425) Sandwich ELISA (Cell Signaling Technology, Frankfurt, Germany) according to the manufacturer’s instructions. In brief, 50 mg of porcine liver tissue or cell pellets (mouse or pig) of one well of a six-well plate were lysed in 500 µL of 1× lysis buffer. Lysates were diluted 1 : 2 by using sample-diluent. Next, 100 µL of diluted samples were incubated overnight at 4 °C in the microwells. After four washing steps (200 µL of 1× wash buffer), 100 µL of detection antibody were added to the wells for 1 h at 37 °C. After another four washing steps, 100 µL of HRP-linked secondary antibody were added for 30 min at 37 °C. Following four washings, 100 µL 3,3', 5,5''-tetramethylbenzidine substrate were added for 10 min. After incubation with 100 µL stop solution, absorbance was measured at 450 nm using the GloMax®-Multi Detection System (Promega, Mannheim, Germany). Relative absorbance was normalized to 100 µg of total protein where indicated. Each sample was run in duplicate in the assay.

3T3-L1 fibroblasts were seeded and differentiated in PLL coated 10 cm dishes or T-175 flasks. Mature adipocytes were stimulated with or without 10 nmol/L human insulin (Sigma-Aldrich) 30 minutes at 37°C. One confluent 10 cm dish per experimental condition was used for the Phospho-Insulin Receptor b (Tyr1150/1151) Sandwich ELISA (Cell Signaling Technology, Leiden, the Netherlands) and one confluent T-175 flask per experimental condition was used for the PIP3 Mass Elisa (Echelon Biosciences, Salt Lake City, UT, USA) were used according to the manufacturer’s protocol.

The relative abundance of pIRβ, pIRS-1 and pIRS-2 in 40 µg of protein isolated from skeletal muscle or liver homogenate was determined using sandwich ELISA (Cell Signaling, Danvers MA) per the manufacturer’s instructions. Spectrophotometric absorbance was read at 450 nm.