Journal: Genes to cells : devoted to molecular & cellular mechanisms

Article Title: AMY-1, a novel C-MYC binding protein that stimulates transcription activity of C-MYC.

doi: 10.1046/j.1365-2443.1998.00206.x

Figure Lengend Snippet: Figure 6 Binding of AMY-1 to C-MYC. The whole length and various segments of the AMY-1 cDNA were expressed in yeast, in bacteria or in mammalian cells and the binding of the proteins to C-MYC was verified. (A) The whole length, the N-terminal half (ES; amino acids 1–63), the C-terminal deletion (EH; amino acids 1–89), the C-terminal half (SX; amino acids 64–104) and the C-terminal end (HX; amino acids 90–104) segments, schematically shown in the figure, were cloned in pGADGH and expressed in the S. cerevisiae HF7C cells (Clontech) transformed with pGBT9-c-myc. After incubation in liquid medium or on filters, b-galactosidase activity was assayed. (B) The wild-type and deletion mutants of AMY-1 fused to thioredoxin (Trx) and (His)6, as well as Trx-(His)6 alone, were expressed in E. coli and subjected to in vitro binding assays. The 1/20 volume of HL60 cell extract used for binding reaction was electrophoresed in the same gel in parallel (lane HL60). (C) The wild-type AMY-1 and the pocket domain of p107 which was fused to GST, as well as GST alone, were expressed in E. coli, purified, and subjected to in vitro binding assay by use of 35S-labelled C-MYC which was synthesized in vitro in reticulocyte lysate. The 1/100 volume of 35S-C-MYC used for the binding reaction was electrophoresed in the same gel in parallel (lane C-MYC). (D) The extract of AM416 cells, constitutively expressing AMY-1-HA, was first immunoprecipitated with an anti-HA antibody (a-HA) or nonspecific mouse IgG (IgG). The precipitates were then separated in a 7.5% polyacrylamide gel containing SDS, blotted on to a filter and reacted with the anti-HA antibody (a-HA) or an anti-C-MYC antibody (a-C-MYC). The 1/5 or 1/20 volume of AM416 cell extract used for binding reaction were electrophoresed in parallel in the same gel (lanes AM416 on left and right, respectively). (E) The extract of HeLa cells was analysed as in (D) using the anti-C-MYC antibody and an anti-AMY-1 antibody (a-AMY-1). The 1/10 or 1/50 volumes of HeLa cell extract used for binding reaction were electrophoresed in parallel in the same gel (left or right of lane HeLa, respectively). (F) The wild-type and various deletion mutants of c- myc were expressed in the yeast cells transformed with pGBT9-AMY-1, and the interaction between myc proteins and AMY-1 was tested as in (A).

Article Snippet: A single band was detected at 64 kDa with a mouse antiC-MYC antibody (C-33, Santa Cruz) in the samples recovered from the resin carrying the Trx-fusion proteins with the full-length (AMY) or the C-terminal half segment of AMY-1 (AMY(SX)), respectively, as well as in the HL60 nuclear extract, but not in the q Blackwell Science Limited Genes to Cells (1998) 3, 549–565 555 samples from the Trx-fused N-terminal half segment of AMY-1 (AMY(ES)) or Trx alone (Fig. 6B).

Techniques: Binding Assay, Bacteria, Clone Assay, Transformation Assay, Incubation, Activity Assay, In Vitro, Synthesized, Expressing, Immunoprecipitation