Effects of CQ on total protein levels of OATP1B1 and Na,K-ATPase in HEK293-OATP1B1 cells and on OATP1B1 total protein levels in human SCH. HEK293-OATP1B1 cells were seeded in 24-well plates at 1.1 × 105 cells/well, and were cultured for 48 h prior to treatment. Human SCH were cultured as described in the Experimental Section. WCL was prepared by directly lysing the cells in the culture plate without trypsinization. Representative immunoblot images of OATP1B1 (A) and Na,K-ATPase (B) in WCL of HEK293-OATP1B1 pretreated with CQ (25 or 100 μM) or CTL for indicated times are shown. GAPDH were used as the loading control for A and B. OATP1B1 and Na,K-ATPase protein levels determined by densitometry were normalized to levels of GAPDH. Fold changes of total protein levels of OATP1B1 in A and Na,K-ATPase in B (CQ vs CTL) are expressed as mean ± SD (n = 4 for both A and B). (C) Immunoblot of OATP1B1 and β-actin in WCL of human SCH pretreated with CQ (10 μM) or CTL for 5 h. OATP1B1 protein levels determined by densitometry were normalized to levels of β-actin. Fold changes of total protein levels of OATP1B1 (CQ vs CTL) are expressed as mean ± SD of n = 3 donor hepatocytes. Representative images are shown.