Darunavir

5-(and-6)-carboxy-2′,7′-dichlorofluorescein (CDCF) and 5-(and-6)-carboxy-2′,7′-dichlorofluorescein diacetate (CDCFDA) were purchased from AAT Bioquest (Sunnyvale, CA, United States). Digoxin, estrone 3-sulfate (E3S), and lopinavir were purchased from Sigma-Aldrich (St. Louis, MO, United States). Nelfinavir was obtained from APExBIO Technology (Houston, TX, United States). Darunavir was purchased from Toronto Research Chemicals (North York, ON, Canada). A rabbit monoclonal P-gp antibody (ab120904), a rabbit monoclonal MRP2 antibody (ab172630) and a rabbit monoclonal GAPDH antibody (ab181602) were purchased from Abcam (Cambridge, MA, United States), and a rabbit monoclonal BCRP antibody (CST42078) was obtained from Cell Signaling Technology (Danvers, MA, United States). All other chemicals and solvents were of the highest grade commercially available.

Lopinavir and darunavir were purchased from Toronto Research Chemicals Inc. (Toronto, Ontario, Canada) and dissolved in ethyl acetate and methanol, respectively. Since the levels of IRS1 expression and IRS1 phosphorylation by insulin were comparable in preliminary experiments, methanol was used as a vehicle control in the following experiments. Insulin from bovine pancreas was obtained from Sigma-Aldrich (St. Louis, MO, USA). The primary antibodies used were anti-phospho (Ser307)-IRS1 and anti-IRS1 antibodies (Upstate Biotechnology Inc., Lake Placid, NY, USA), and anti-SOCS1, anti-SOCS3 and anti-PTP1B antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The phospho-tyrosine-specific monoclonal antibody 4G10 (Upstate Biotechnology, Darmstadt, Germany) was used in the immunoprecipitation assay.