Figure 1. ISWI is a novel... | Rockefeller University Press

$Figure 1. ISWI is a novel RanGTP-dependent MAP bearing NLS. (A) Preparation of MAPs from the NLS protein fraction and identification of ISWI in the MAP fraction. The NLS protein fraction (NLS) was incubated with taxol-stabilized pure MTs. The MTs were sedimented and MAPs were eluted with 500 mM KCl. The eluate was resolved on SDS-PAGE for silver staining (top) or immunoblotting with anti-hSNF2H antibody (bottom). (B) Behavior of ISWI during sequential preparation of NLS proteins and MAPs. (Top) To isolate NLS proteins, a CSF extract was treated with RanQ69L beads, the supernatant (activated extract) was further incubated with importin-β beads, and the subsequent supernatant (depleted extract) was recovered. Each extract was blotted with anti-hSNF2H antibody. Tubulin was used as a loading control. (Bottom) To isolate MAPs, the NLS protein fraction (NLS) was incubated with taxol-stabilized MTs and then centrifuged to separate the flow-through (FT) and the MT pellet. The pellet was incubated with 500 mM NaCl and centrifuged again. The supernatant is a fraction containing MAPs and the pellet is the MTs after elution. Each fraction was immunoblotted for ISWI. (C) Recombinant ISWI expressed in insect cells and purified on Talon beads and a Mono S column. Coomassie-stained gel. (D) ISWI directly binds MTs in vitro. Recombinant ISWI (0.25 µM) was incubated with or without taxol-stabilized pure MTs (1 µM) in BRB80 buffer. After centrifugation, supernatant (s) and pellet (p) were resolved on SDS-PAGE for Coomassie staining (top) or immunoblot (bottom). BSA was used as a carrier protein and a negative control that does not bind MTs. (E) Regulation of ISWI binding to MTs by RanGTP and importin-α/β. Recombinant ISWI (0.25 µM) was incubated with 2 µM taxol-stabilized MTs in CSF-XB buffer in the presence or absence of 1 µM importin-α, 1 µM ED mutant, 1 µM importin-β, and 5 µM RanQ69L. After centrifugation, the supernatant (s) and pellet (p) were analyzed by immunoblot using anti-hSNF2H antibody.$

Figure 1.

ISWI is a novel RanGTP-dependent MAP bearing NLS. (A) Preparation of MAPs from the NLS protein fraction and identification of ISWI in the MAP fraction. The NLS protein fraction (NLS) was incubated with taxol-stabilized pure MTs. The MTs were sedimented and MAPs were eluted with 500 mM KCl. The eluate was resolved on SDS-PAGE for silver staining (top) or immunoblotting with anti-hSNF2H antibody (bottom). (B) Behavior of ISWI during sequential preparation of NLS proteins and MAPs. (Top) To isolate NLS proteins, a CSF extract was treated with RanQ69L beads, the supernatant (activated extract) was further incubated with importin-β beads, and the subsequent supernatant (depleted extract) was recovered. Each extract was blotted with anti-hSNF2H antibody. Tubulin was used as a loading control. (Bottom) To isolate MAPs, the NLS protein fraction (NLS) was incubated with taxol-stabilized MTs and then centrifuged to separate the flow-through (FT) and the MT pellet. The pellet was incubated with 500 mM NaCl and centrifuged again. The supernatant is a fraction containing MAPs and the pellet is the MTs after elution. Each fraction was immunoblotted for ISWI. (C) Recombinant ISWI expressed in insect cells and purified on Talon beads and a Mono S column. Coomassie-stained gel. (D) ISWI directly binds MTs in vitro. Recombinant ISWI (0.25 µM) was incubated with or without taxol-stabilized pure MTs (1 µM) in BRB80 buffer. After centrifugation, supernatant (s) and pellet (p) were resolved on SDS-PAGE for Coomassie staining (top) or immunoblot (bottom). BSA was used as a carrier protein and a negative control that does not bind MTs. (E) Regulation of ISWI binding to MTs by RanGTP and importin-α/β. Recombinant ISWI (0.25 µM) was incubated with 2 µM taxol-stabilized MTs in CSF-XB buffer in the presence or absence of 1 µM importin-α, 1 µM ED mutant, 1 µM importin-β, and 5 µM RanQ69L. After centrifugation, the supernatant (s) and pellet (p) were analyzed by immunoblot using anti-hSNF2H antibody.

This Feature Is Available To Subscribers Only