Figure 4

Y-box binding protein 1 (YB-1) binds to the epidermal growth factor receptor (EGFR) promoter. (a) Chromatin immunoprecipitation was performed on SUM149 cells. YB-1 binds to the EGFR promoter in the basal-like cells where the 2a loci is the preferred binding site (lane 2). Weak binding was also detected with the 1b primers (lane 1). No binding was observed in the 2b or 3 sites (lanes 3 to 4), nor was there any non-specific binding detected in the IgY negative controls (lanes 5 to 8). Input DNA was diluted fourfold and amplified to demonstrate that the primer produced an expected product (lanes 9 to 12). The no input controls (lanes 13 to 16) are presented to show a lack of non-specific amplifications. (b) Serial ChIP was performed by sequentially pulling down YB-1 and then immunoprecipitating with a phospho-serine antibody. This demonstrated that at least some of the YB-1 is serine phosphorylated when bound to the EGFR 2a site. YB-1 binds to the 2a site (lane 1) as expected. Similarly, the phospho-serine antibody pulls down a complex that can be amplified with the 2a primers (lane 2). Re-ChIP with the YB-1 antibody and subsequently with the phospho-serine antibody also bound to EGFR at the 2a site (lane 3). A phospho-serine YB-1 complex bound to the 2a site on EGFR (lane 3). Species-matched IgG and IgY controls were included to show that the binding was specific (lane 4). The input DNA and no DNA controls were also included (lanes 5 and 6). (c) ChIP was carried out using a phospho-YB-1 antibody (S102), and binding was detected for the EGFR 2a region (lane 4). There was no binding observed when immunoprecipitation was performed using IgG as a control (lane 3). Input DNA was diluted fourfold and amplified to demonstrate that the primer produced an expected product (lanes 5 and 6). Lane 1 is the DNA ladder.