|
|
|
|
|
|
|
|
|||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | |||||
|
Fig. 2. NoLS interacts directly with B23; this interaction involves the acidic stretches of B23. (A) GST and GST-NoLS pull-down assays performed using whole HeLa cell extracts. A sample (1%) of the extract used for each pull-down assay (Input), 1% of the supernatant (S) and 1.5% of the proteins eluted from the bead pellet (P) after the indicated pull-down assay were electrophoresed and immunoblotted to detect B23, Nopp140, Nucleolin and Nop52. B23 interacts specifically with GST-NoLS but not with GST. By contrast, Nopp140, Nucleolin and Nop52 do not interact with GST-NoLS. (B) GST and GST-NoLS pull-down assays performed using a purified recombinant B23. A sample (2%) of the supernatant (S) and 3% of the proteins eluted from the bead pellet (P) after the indicated pull-down assay were electrophoresed and immunoblotted to detect B23. B23-NoLS interaction is direct. (C) Pull-down assays performed on whole extracts prepared from HeLa cells transiently transfected with B23 wild-type (B23 wt) and B23 mutants fused to the C-terminus of GFP or DsRed as indicated. The mutants B23/121-186, B23/1-119, B23/1-132, B23/1-160, B23/1-186 and B23/187-294 correspond to the B23 mutants possessing the B23 residues 121-186, 1-119, 1-132, 1-160, 1-186 and 187-294, respectively. Black boxes on the schematic representations correspond to the stretches of acidic amino acids present in the B23 sequence. A sample (1.5%) of the extracts used for each pull-down assay (Input) and 3% of the proteins eluted after the GST-NoLS pull-down assay (GST-NoLS) were electrophoresed and immunoblotted using anti-B23 or anti-GFP antibodies. Only B23 mutants possessing at least one stretch of acidic amino acids interact with GST-NoLS. Asterisks represent the endogenous B23 revealed only with the anti-B23 antibodies but not with the anti-GFP antibodies. Arrowheads point to the overexpressed tagged-B23 or mutant B23.
|
