Rabbit anti na k atpase

LX2 cells (kindly provided by Prof. Dr. S. Friedman) were cultured in DEME media containing 10 % (v/v) FBS (Gibco, USA) and penicillin/streptomycin (100 ug/ml, Invitrogen, Carlsbad, CA) at 37 °C in 5 % CO₂. Membrane and cytosolic fractions were extracted from LX2 cells using a plasma membrane protein extraction kit (Abcam, Cambridge, MA). The expression of FGL2 was assessed by Western blot as previously described [23 (link)]. Membranes were first incubated with rabbit anti-Na +/K + ATPase (1:1,000, Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-Fgl2 (1:400; Abnova, Taiwan, China) and mouse anti-β-actin (1:3,000; Sigma, St Louis, MO, USA) primary antibodies and subsequently with either goat anti-mouse (1:2,500; Bio-Rad Laboratories, Veenendaal, The Netherlands) or goat anti-rabbit (1:5,000; Santa Cruz Biotechnology, CA, USA)-horseradish peroxidase conjugated secondary antibodies. Membranes were incubated with ECL-Plus reagent (Amersham, Piscataway, NJ), and chemiluminescence was detected using BioMax MR Film (Kodak, Rochester, NY).

Relative levels of wild-type and mutant MRP1 in WCE and membrane vesicles were determined by immunoblot analysis as before [54 (link)] using mouse mAb QCRL-1 anti-human MRP1 (diluted 1:10,000) (epitope amino acids ⁹¹⁸SSYSGDI⁹²⁴ [54 (link)]) and mouse anti-α-tubulin (diluted 1:10,000) (Sigma T6074) or rabbit anti-Na⁺/K⁺-ATPase (diluted 1:2,500) (Santa Cruz Biotechnology 28800) as a protein loading control followed by incubation with horseradish peroxidase-conjugated goat anti-mouse (ThermoFisher Scientific 31430) or anti-rabbit (Cedarlane CLAS10-667) antibodies (diluted 1:10,000), respectively. Antibodies bound to the blot were detected using a Western Blotting Chemiluminescence Reagent Plus Kit (PerkinElmer NEL105) and the blot exposed to HyBlot CL autoradiography film (Denville Scientific) for several time periods. Signals on the film were quantified by densitometry using ImageJ software [55 ] and values adjusted if needed, according to the signal of the protein loading control. Mutant MRP1 levels were then expressed relative to wild-type MRP1 levels and data analysed for differences using GraphPad Prism^TM (GraphPad Software, La Jolla, CA) using an unpaired t-test with a significance threshold of P<0.05.