*4.4. Immunoblot Analysis of Wild-Type and Mutant rAbcc6*

The expression of rAbcc6 was confirmed by immunoblot analysis as described previously [19]. Briefly, cell lysates were prepared in lysis buffer (0.1% Triton-x-100, 10 mM KCl, 10 mM Tris-HCl and 1.5 mM MgCl2, pH 7.4) supplemented with protease inhibitors (EDTAfree Protease Inhibitor Cocktail, Sigma Aldrich, St. Louis, MO, USA. Samples containing

5 µg of total protein determined by BCA assay were separated on 7.5% SDS-polyacrylamide gels (Bio-Rad, Hercules, CA, USA and transferred to a PVDF membranes using a semi-dry blotting system (Trans-Blot Turbo, Bio-Rad, Hercules, Ca, USA). rAbcc6 was detected with the polyclonal K14 rabbit anti-rAbcc6 antibody (diluted 1:3000) (kind gift of Dr. Bruno Stieger) and horseradish peroxidase (HRP)-conjugated donkey anti-rabbit secondary antibody (1:5000) (Fisher Scientific, Waltham, MA, USA). Mouse anti-α-tubulin (1:1000) (Sc-23948, Santa-Cruz Biotechnology, Dallas, TX, USA) followed by HRP-conjugated polyclonal rabbit anti-mouse IgG employed as secondary antibody (1:5000) (P0161, Dako, Agilent, Santa Clara, CA, USA), was used as a protein loading control. Antibody binding was visualized by ECL (Pierce Western blotting substrate, Thermo Scientific, Waltham, MA, USA).
