Heterologous expression of AtMRP6 in yeast and mammalian cells. (A) Immunodetection of GFP by western-blot analysis on total yeast proteins extracted by the trichloroacetic acid method. AtMRP6-GFP and YCF1-GFP lanes represent proteins extracted from yeast cells transformed by pYES2 AtMRP6-GFP and pYES2 YCF1-GFP, respectively. YCF1-GFP (165 kDa) was used as a positive control. Only the C-terminal part of AtMRP6 was preserved as a polypeptide of an apparent molecular mass of 81 kDa (theorical mass with the GFP: 192 kDa). (B) Immunodetection of GFP by western-blot analysis of HEK-293 cell proteins extracted by the RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% triton, antiproteases coktail). Empty-vector and AtMRP6-GFP lanes represent total proteins extracted from HEK-293 cells transfected by jetPEI with pCi and pCi AtMRP6-GFP, respectively. (C) Corresponding cells expressing AtMRP6-GFP in HEK-293 cells observed under fluorescence microscopy (excitation was performed at 488 nm, emission collected at 510 nm). As a control, cells expressing only GFP (pEGFP-N2) are presented in the lower panel.