## 3 Results
### Identification of IGF-IR Promoter-Binding Transcription Factors by Western Blots  
Western blot analysis of DNA affinity chromatography-purified proteins identified a number of previously reported as well as novel IGF-IR promoter binding proteins. The data are summarized in Figure 4 and Tables 3 and 4. Consistent with their role as positive regulators of the IGF-IR gene, the binding of zinc finger proteins Sp1 and KLF6 to the IGF-IR promoter was stronger in MCF7 than in C4.12.5 cells. Similarly, ER-\(\alpha\Box\) binding to the IGF-IR promoter was stronger in MCF7 than in C4.12.5 cells. On the other hand, a number of transcription factors, including AR, Smad2/3, E2F1, c-jun, TCF1A, TCF3A, GSK3B, and p21 appear to bind to the promoter region only in the C4.12.5 cell line. p53, c-fos, and PARP bind to IGF-IR promoter elements in both cell lines with similar affinity. No DNA binding was detected in either cell line using antibodies against BRCA1, TOPOII, IRS-1, MET, p120, Foxo1, p73, lamin A/C, c-Myc, p63, cyclin D, cyclin G, histone1, and BRCA2. \begin{table}
\begin{tabular}{|p{113.8 pt}|p{113.8 pt}|p{113.8 pt}|} \hline Mucin & Cytoplasm Mitochondria Nucleus & Activation of Wnt target genes. \\ \hline Nucleoporin 35 [Nuclear pore glycoprotein-210 (gp210)] & Membrane Cytoplasm Nucleus & Trafficking regulator in the eukaryotic nuclear pore complex. \\ \hline p80 protein & Cytoplasm Nucleus & Part of the nuclear coiled bodies (CBS) involved in assembly/disassembly of nucleoplasmic snRNPs. \\ \hline Paraspeckle component 1 & Cytoplasm Nucleus Nucleus & Regulates cooperatively with NONO and SFPQ, androgen receptor-mediated gene transcription activity. \\ \hline PREDICTED: similar to RAN protein Nucleus & Cytoplasm Nucleus & Inhibits GTP exchange on Ran. \\ \hline Regulator of chromosome condensation (RCC1) Nucleus & Cytoplasm Nucleus & Promotes the exchange of Ran-bound GDP by GTP. \\ \hline Retinoblastoma binding protein 7 & Nucleus Nucleus & Modulates the functions ascribed to BRCA1 in transcriptional regulation, DNA repair, and/or cell cycle checkpoint control. \\ \hline SERPINE1 mRNA binding protein 1 isoform 1 Nucleus & Cytoplasm Nucleus & Regulates mRNA stability. \\ \hline SPIN & Nucleus Nucleus & Regulation of cell cycle \\ \hline Sterile alpha motif domain containing 1 & Nucleus Nucleus Proteins. & Widespread domain in signalling and nuclear proteins. \\ \hline Thymopoietin \(\gamma\) and \(\beta\) ( TMPOs) Nucleus Nucleus Transcription factor A, mitochondrial Mitochondria Nucleoval in mitochondrial transcription regulation. \\ \hline UV excision repair protein RAD23 homolog B Nucleus & Cytoplasm Nucleus & Plays a role both in proteasomal degradation of misfolded proteins and DNA repair. \\ \hline \end{tabular}
\end{table} 
Table 2: _Cont._
Figure 4: IGF-IR promoter-binding transcription factors identified by Western blots in the MCF7 and C4.12.5 cell lines. DNA chromatography eluates were electrophoresed though 10% SDS-PAGE and blotted with the antibodies listed under _Materials and Methods_ Section. The left lanes correspond to nuclear extracts (NE; 4.2 \(\upmu\)g) and the right lanes correspond to DNA affinity chromatography eluates (E; 10 \(\upmu\)g). \begin{table}
\begin{tabular}{l l l l} \hline
**Gene** & **MCF7** & **C4.12.5** & **Nomenclature** \\ \hline BRCA1 & 1 & 0.3 & Breast and ovarian cancer type I \\ TOPOII & 1 & 0 & Type II topoisomerase \\ IRS-I & 0 & 0 & Insulin receptor substrate-1 \\ PARP & 1 & 2 & Poly ADP ribose polymerase \\ MET & 1 & 0 & Met tyrosine kinase \\ p120 & 1 & 1.39 & Zinc finger protein 120 \\ AR & 1 & 0.64 & Androgen receptor \\ Sp1 & 1 & 0.79 & Trans-acting transcription factor 1 \\ Foxo1 & 1 & 0.5 & Forkhead Box A1 \\ \hline \end{tabular}
\end{table} 
Table 3: Differentially expressed nuclear proteins identified by Westen blots in MCF7 and C4.12.5 cells.