Bcl-3 Expression and Nuclear Translocation Are Induced by Granulocyte-Macrophage Colony-Stimulating Factor and Erythropoietin in Proliferating Human Erythroid Precursors by Min-Ying Zhang, Edward W. Harhaj, Laurie Bell, Shao-Cong Sun, and Barbara A. Miller Blood Volume 92(4):1225-1234 August 15, 1998 ©1998 by American Society of Hematology

Bcl-3 expression in GM-CSF–stimulated and Epo-stimulated TF-1 cells. Bcl-3 expression in GM-CSF–stimulated and Epo-stimulated TF-1 cells. Whole cell lysates were prepared from growth factor-induced TF-1 cells at the time points indicated. Thirty micrograms of protein was loaded on each lane of a 12% polyacrylamide gel (Bcl-3) or 15 μg of protein was loaded on each lane of a 10% polyacrylamide gel (IκB). The membranes were blotted with anti–Bcl-3 (1:200) or anti-IκB (1:2,000) antibody as a control and then detected with ECL. N.D. indicates time points that were not done. Three independent experiments showed induction of Bcl-3 expression. Min-Ying Zhang et al. Blood 1998;92:1225-1234 ©1998 by American Society of Hematology

GM-CSF and Epo induce Bcl-3 mRNA expression in TF-1 cells. GM-CSF and Epo induce Bcl-3 mRNA expression in TF-1 cells. Total RNA was isolated from GM-CSF–induced or Epo-induced TF-1 cells and Northern blotting analysis was performed using 40 μg RNA from each sample. 32P-dCTP–labeled Bcl-3 cDNA was used as a probe. 18s rRNA is shown as a control for equivalent loading. Min-Ying Zhang et al. Blood 1998;92:1225-1234 ©1998 by American Society of Hematology

GM-CSF and Epo enhance Bcl-3 nuclear translocation. GM-CSF and Epo enhance Bcl-3 nuclear translocation. Growth factor-induced TF-1 cells (1 × 107) were used for nuclear or cytoplasm separation. Twenty-five micrograms of nuclear or 20 μg of cytosolic protein extracts was loaded on each lane of a 12% polyacrylamide gel and subjected to Western blotting with anti–Bcl-3 antibody. As a control, 15 μg of nuclear or cytoplasmic extract was loaded on a 10% polyacrylamide gel to detect IκB. For GM-CSF–stimulated cells, 30 μg of nuclear or cytoplasmic extract was loaded on each lane of 12% gel and detection was with anti-E47 antibody as another control for quality of subcellular fractionation. Representative results are shown from 3 independent experiments. Min-Ying Zhang et al. Blood 1998;92:1225-1234 ©1998 by American Society of Hematology

Bcl-3 expression in day-7, -10, and -14 BFU-E–derived erythroblasts. Bcl-3 expression in day-7, -10, and -14 BFU-E–derived erythroblasts. (A) Normal human BFU-E–derived erythroblasts were harvested and the whole cell lysates from 4 × 105 (Bcl-3) or 2 × 105 (IκB) cells were separated on a 10% polyacrylamide gel. Western analysis was performed with anti–Bcl-3 or anti-IκB antibodies and ECL. (B) Nuclear and cytoplasmic extracts were separated from day-10 cells. Fifty micrograms of nuclear (N) or cytoplasmic (C) extract was loaded onto each lane of a 10% gel and subjected to Western blotting. Two experiments were performed with anti–Bcl-3 or anti-IκB antibodies with identical results. Min-Ying Zhang et al. Blood 1998;92:1225-1234 ©1998 by American Society of Hematology

Bcl-3 is expressed in many human tissues. Bcl-3 is expressed in many human tissues. Protein extracts from normal human tissues were prepared as described in the Materials and Methods. Thirty micrograms of protein was loaded on each lane of a 10% polyacrylamide gel and detection was with anti–Bcl-3 antibody. Min-Ying Zhang et al. Blood 1998;92:1225-1234 ©1998 by American Society of Hematology

Bcl-3 is hyperphosphorylated in TF-1 cells and BFU-E–derived erythroblasts. Bcl-3 is hyperphosphorylated in TF-1 cells and BFU-E–derived erythroblasts. (Top) Whole cell lysates (W) and nuclear (N) and cytoplasmic (C) extracts were prepared from Epo-induced or GM-CSF–induced TF-1 cells. Thirty micrograms of each extract was incubated with or without 26 U of CIP at 37°C for 40 minutes and then subjected to Western blotting assay with anti–Bcl-3 antibody and ECL. (Bottom) Thirty micrograms of whole cell lysate (W) from normal human heart tissue or 50 μg from day-10 BFU-E–derived erythroblasts was also incubated with or without 26 U CIP and subjected to Western blotting with anti–Bcl-3 as described in the Materials and Methods. Three experiments were performed with similar results. (+) with CIP; (−) without CIP. Min-Ying Zhang et al. Blood 1998;92:1225-1234 ©1998 by American Society of Hematology

Activation of an HIV-1 κB-TATA-luciferase reporter plasmid after overexpression of Bcl-3 in TF-1 cells. Activation of an HIV-1 κB-TATA-luciferase reporter plasmid after overexpression of Bcl-3 in TF-1 cells. A total of 0.5 μg of plasmids expressing Bcl-3, p50, or p52 was cotransfected with 0.5 μg of the HIV-1 κB-TATA-luciferase reporter plasmid into TF-1 cells separately or in combination. The pGL2 basic vector (0.5 μg) was used as negative control. Where noted, 1.0 μg of Bcl-3 was cotransfected. The total amount of transfected DNA was kept constant by adding appropriate amounts of expression vector without insert. At 48 hours after transfection, cells were collected for luciferase assay. Results are expressed as the mean ± SEM (×103 cpm). Five experiments were performed. *A significant increase above the κB-TATA reporter plasmid (P ≤ .05). Min-Ying Zhang et al. Blood 1998;92:1225-1234 ©1998 by American Society of Hematology

Bcl-3 is associated with NF-κB p52 in TF-1 cells (EMSA). Bcl-3 is associated with NF-κB p52 in TF-1 cells (EMSA). (A) Six micrograms of nuclear extracts prepared from TF-1 cells stimulated with GM-CSF for 24 hours was incubated with different NF-κB antibodies for 10 minutes before adding a32P-labeled c-myb κB binding oligonuclear probe. Three DNA-protein complexes were generated. Complexes 2 and 3 (C2 and C3) were supershifted by anti-p50; C1, C2, and C3 were shifted by anti-p52. C1 was reproducibly inhibited by anti–Bcl-3 antibody (with a long exposure, a supershifted band was also visible). However, anti-RelB and anti–c-Rel had no effect on any of these complexes. Similar results were observed in 3 experiments. (B) EMSA was performed with growth factor-deprived TF-1 cells or TF-1 cells induced with GM-CSF for 24 hours. The C1 complex was greatly increased by GM-CSF stimulation, whereas other complexes had no significant change. This experiment was repeated 3 times with similar results. Min-Ying Zhang et al. Blood 1998;92:1225-1234 ©1998 by American Society of Hematology

Overexpression of Bcl-3 and p50 or p52 activates ac-myb κB-TATA-luciferase reporter plasmid. Overexpression of Bcl-3 and p50 or p52 activates ac-myb κB-TATA-luciferase reporter plasmid. A total of 0.5 μg of plasmids expressing Bcl-3, p50, or p52 was cotransfected with 1.0 μg of c-myb κB-TATA-luciferase reporter plasmid into TF-1 cells separately or in combination. The pGL2 basic vector was used as negative control. The total amount of transfected DNA (2 μg) was kept constant by adding appropriate amounts of expression vector without insert. At 48 hours after transfection, cells were collected for luciferase assay. Results are expressed as the mean ± SEM (×103 cpm). Three experiments were performed. *A significant increase above the c-myb κB-TATA reporter plasmid (P < .05). Min-Ying Zhang et al. Blood 1998;92:1225-1234 ©1998 by American Society of Hematology

Deoxyhemoglobin S crystal. Deoxyhemoglobin S crystal. Chromatographically purified HbS (in 5 mmol/L KCl, 10 mmol/L Tris, pH 6.5) was deoxygenated, air-dried onto a graphite chip under a stream of nitrogen, and examined by scanning tunneling electron microscopy (STEM). Images at two levels of resolution are shown for a portion of a single crystalline bundle of HbS fibers that was 1,450 nm long and 65 nm in diameter. The size of the individual constituent subunits (6 × 5 nm) is consistent with their identity as hemoglobin tetramers. The vivid detail at the interface between tetramers (asterisk) suggests that STEM could be used to help define the fine structure of HbS polymers. (Courtesy of Mary M. Christopher, Yi Lin, Roy Matthew, D. Fennell Evans, and Robert P. Hebbel, Departments of Medicine and Chemical Engineering, University of Minnesota, Minneapolis, MN 55455.)‏ Min-Ying Zhang et al. Blood 1998;92:1225-1234 ©1998 by American Society of Hematology