Figure 3.

GR binds to importin 7 and importin 8. Input lane corresponds to 20% input. (A) GST, GST-importin 7 (7), GST-importin 8 (8), or GST-importin α (α) (4 μg) were immobilized on glutathione resin and incubated with 150 ng of purified rat GR for 2 h at 4°C. Bound proteins were resolved by SDS-PAGE and immunoblotted with rat GR-specific BuGR2 antibody. (B) Immobilized GST proteins in A were incubated with 12 ng in vitro transcribed and translated N795, N525, or 540C for 1 h at RT. Bound proteins were resolved by SDS-PAGE and detected by PhosphorImager. (C) Immobilized GST-BIB, GST NLS, GST-407-525, GST-407-525 K513-515A, or GST alone (4 μg) were incubated with 4 μg of purified importin 7 overnight at 4°C. Bound proteins were resolved by SDS-PAGE and immunoblotted with a polyclonal antibody to importin 7. (D) Substrates described in C were incubated with 72 μg of HeLa extract overnight at 4°C and immunoblotted as above. (E) Immobilized import receptors (4 μg) were preincubated with 40 μM RanQ69L for 1 h at 4°C and then incubated with 12 ng in vitro transcribed and translated full-length GR for 2 h at 4°C. (F) GST, GST-α, and GST-7 (4 μg) were immobilized on glutathione resin in the presence or absence of importin β overnight at 4°C. Bound import receptor complexes were incubated with 4 μg of a purified GR fragment (407–565) containing NL1 for 3 h at 4°C.