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Figure 7 | BMC Molecular Biology

Figure 7

From: Chibby forms a homodimer through a heptad repeat of leucine residues in its C-terminal coiled-coil motif

Figure 7

Homodimer formation of Cby is a prerequisite for its efficient nuclear import. (A) C-terminally Flag-tagged CbyL77A/L91A accumulates in the cytoplasm. COS7 cells were transiently transfected with an expression vector encoding C-terminally Flag-tagged WT or mutant Cby as indicated. Cells were fixed 24 hr post-transfection, followed by immunostaining with anti-Flag antibody. Nuclei were stained with DAPI. (B) A fraction of N-terminally Flag-tagged CbyL77A/L91A is found in the cytoplasm after LMB treatment. COS7 cells were transfected with an expression vector for N-terminally Flag-tagged CbyWT or CbyL77A/L91A, treated with methanol (- LMB) or 40 nM LMB (+ LMB) for 5 hr, and immunostained with anti-Flag antibody for Cby. Nuclei were counterstained with DAPI. (C) Quantitative analysis of the results in (B). The subcellular localization of Flag-CbyWT and Flag-CbyL77A/L91A was scored as follows: N>C, predominantly nuclear; N = C, evenly distributed between the nucleus and cytoplasm; N<C, predominantly cytoplasmic. Error bars represent the means ± SD of three independent experiments. (D) Reduced binding of CbyL77A/L91A to importin-α. Bacterially produced MBP or the indicated MBP-Cby protein was incubated with GST-importin-α3. The complexes were then pulled down with amylose resin and subjected to Western blotting using anti-GST antibody (top panel). The input lane was loaded with one-fiftieth of the amount of GST-importin-α3 used in the binding reactions (lane 1). One-thirtieth of each pull-down sample was run on a separate SDS-PAGE and immunoblotted with anti-MBP antibody, showing that similar amounts of MBP-Cby protein were pulled down (bottom panel).

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