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. 2010 Sep 16;285(48):37314–37323. doi: 10.1074/jbc.M110.180786

FIGURE 4.

FIGURE 4.

Effect of knockdown of importin β1 on the subcellular localization of EGFP-cNLS and Mycd in cultured VSMCs. A, VSMCs were transfected with the indicated siRNAs and cultured for 2 days. Whole-cell extracts were then subjected to immunoblotting using the indicated antibodies. The level of α-tubulin served as a loading control. B–D, VSMCs were first transfected with the indicated siRNAs and cultured for 24 h. They were then transfected with the expression plasmid for FLAG-tagged EGFP-cNLS (B) or FLAG-tagged Mycd (C) and cultured for a further 24 h. The cells were stained with an anti-FLAG antibody (green (B) or red (C)) and Hoechst 33258 (blue), and the data in the images were quantified as described under “Experimental Procedures.” Representative images are shown (B and C). Bars = 10 μm. D, VSMCs were transfected with the indicated siRNAs and cultured for 45 h and then further cultured for 3.5 h with vehicle or MG-132 (5 μm). Their cytoplasmic (C) and nuclear (N) fractions were analyzed by immunoblotting using the indicated antibodies. Representative results from at least three independent experiments are shown. NC, diffuse distribution in the nucleus and the cytoplasm.