FIGURE 8.
Association of karyopherins/importins and Nrf2 as revealed by their co-immunoprecipitation. K562 cells (4 × 106/8 ml of medium in T25 flask) were cultured for 24 h followed by the addition of tBHQ (20 μm). The cells were harvested at the indicated time points (up to 60 min) after the addition of tBHQ. Nuclear and cytoplasmic extracts were prepared as described previously (27, 43). A, representative blots of immunoprecipitates generated with antibody against karyopherin α1 (importin α5) or karyopherin β1 (importin β1) followed by Western blotting for Nrf2. Immunoprecipitation was performed as described under “Experimental Procedures” using nuclear extract or cytoplasmic fraction (20 μg of protein in each case) and 2 μg each of anti-karyopherin α1 (importin α5) antibody (sc-6918, Santa Cruz Biotechnology, Inc.), anti-karyopherin β1 (importin β1) antibody (sc-11367, Santa Cruz Biotechnology, Inc.), or normal rabbit IgG. Immunoprecipitated material (corresponding to 5–10 μg of protein from the nuclear extract or cytoplasmic fraction) was analyzed by Western blotting using anti-Nrf2 antibody (sc-13032, Santa Cruz Biotechnology, Inc.). Upper panel, lane 1, normal IgG (control); lanes 2–5, immunoprecipitates with antibody against karyopherin α1 (importin α5) or karyopherin β1 (importin β1) at 0, 15, 30, and 60 min after addition of tBHQ. Lower panel, graphic representation of the data in the upper panel. Quantitation of the density of bands representing co-immunoprecipitated Nrf2 was assessed by densitometric scanning and expressed relative to the band at zero time (no tBHQ), which was set as 100%. Data are means ± S.E. for three different experiments. B, effect of SN50 on the association of Nrf2 with importin α. K562 cells were pretreated for 1 h with or without 10 or 25 μm SN50 or the control peptide SN50M followed by incubation with tBHQ (20 μm) for 1 h. Nuclear extracts were then prepared and used for co-immunoprecipitation analysis as in A. IP, immunoprecipitate; WB, Western blot; Cyto, cytoplasmic extract; Ctrl, control; nuclear, nuclear extract.