SATB1 increases hemoglobin expression. (A) Western blotting for K562 cells and K562/SATB1 (K/SATB) cells using anti-SATB1 antibody with β-actin (βac) as a loading control and corresponding cell pellets is shown. (B) Benzidine staining for K562 and K562/SATB1 (K/SATB) cells is shown. (Microscope: Olympus 1X70 [Melville, NY], 100 × magnification; cells in PBS; Spot camera [Spot Diagnostic Instruments, Sterling Heights, MI] with Spot 3.02 application software.) (C) Hemoglobin production (pg/cell) in stable K562/SATB1 clones is plotted versus the protein level determined by Western blotting with anti-SATB1 antibody (SATB) and normalized to β-actin. (D) K562 cell mRNA expression was determined for ϵ-globin, γ-globin, and β-globin as indicated (left panel) and normalized to β-actin. Globin gene expression was also determined for K562/SATB1 cells (▪) and cells stably transfected with a vector control (□; right panels as indicated). (E) For K562/SATB1 clones, ϵ-globin expression normalized to β-actin is plotted versus the protein level determined by Western blotting with anti-SATB1 antibody (SATB) normalized to β-actin. (F) Western blotting shows the expression of GATA-1 (G1) and GATA-2 (G2) in K562 and K562/SATB1 cells and in cells stably transfected with the control vector. Loading controls are β-tubulin (βtu) and β-actin (βac). (G) A GATA-1 DNA probe and nuclear extracts from K562 cells (lanes 1-7, 11), and from K562/SATB1 clones no. 3 (lane 8), no. 10 (lane 9), and no. 17 (lane 10) expressing 3- to 4-fold increase in SATB1 were used for EMSA. GATA-1 binding was competed by DNA containing a GATA-1 binding motif (G; lane 2) with increasing amounts of competitor (lane 6, 60 ×; lane 7, 120 ×) and anti-GATA-1 antibody (αG; lane 4) but not by a mutated GATA-1 motif (ΔG; lane 3). NC indicates no specific competitor added. Error bars represent SD from 3 independent measurements.