SALL4 Affects the Side Population Figure 4. SALL4 binds to the promoter regions of ABCA3. (A) ChIP-qPCR showed that SALL4 could bind to ABCA3 but not ABCG2 promoter. Antibody against SALL4 was used to immunoprecipitate DNA fragments from SALL4B-overexpressed 293 cells and fold enrichment was compared to input after normalization with GAPDH. Three pairs of primers in the ABCA3 promoter region (left panel) and two pairs of primers in the ABCG2 promoter region (right panel) were used in ChIP-qPCR analysis. The ABCA3 promoter was found to be bound by SALL4B. (B), For EMSA, Western blot analysis using anti-SALL4 antibody was performed on whole cell lysate (left) or nuclear extract (right) from HeLa cells expressing SALL4B. (C), EMSA probes sequences (underlined, bold) in the ABCA3 promoter region used for EMSA are indicated. EMSAs were performed to analyze the binding of proteins from nuclear extract of SALL4B expressed HeLa cells to the ABCA3 promoter as described under ‘‘Materials and Methods’’. (D), EMSA using three different probes in the ABCA3 promoter regions. Lane 1–3 are with probe #1, lane 4–6 are with probe #2, and lane 7–9 are with probe #3. Lane 1, 4 and 7 are probe-only reactions, a 200-fold excess of unlabelled probes of each probe was added in lane 3, 6 and 9. (E), EMSA was performed with probe #1. Lane 1, labeled oligonucleotide; lane 2, labeled oligonucleotide + nuclear extract; lane 3, labeled oligonucleotide + nuclear extract + 200-fold molar excess of unlabeled oligonucleotides; lane 4, labeled oligonucleotide + nuclear extract + anti-SALL4 antibody; lane 5, labeled oligonucleotide + nuclear extract + anti-Tubulin antibody. Rabbit IgG (lane 6) and mouse IgG (lane 7) were used as controls. Results denote mean 6 S.D. from two independent experiments. Y axis: relative fold enrichment of ChIP-promoter region in SALL4B pull-downs when compared to that of input. doi:10.1371/journal.pone.0018372.g004 PLoS ONE | www.plosone.org 7 April 2011 | Volume 6 | Issue 4 | e18372