1. by Madhu Gupta, Paul T. Mungai, and Eugene Goldwasser
A new transacting factor that modulates hypoxia-induced expression of the erythropoietin gene
by Madhu Gupta, Paul T. Mungai, and Eugene Goldwasser
Blood
Volume 96(2):
July 15, 2000
©2000 by American Society of Hematology

2. Gel-shift assay with HAF protein
Gel-shift assay with HAF protein.The probe used was the region −45 to −61 bp (EP17) of the epo promoter.
Gel-shift assay with HAF protein.The probe used was the region −45 to −61 bp (EP17) of the epo promoter. (A) Gel-shift assay using a crude extract from HAF recombinant lysogen. The crude extract (CE) (5 μg) was incubated with radiolabel led EP17 alone (lane 1), or in the presence of a 500-fold molar excess of the specific competitor EP31 (lane 2) or nonspecific competitor EP22 (lane 3). (B) Gel-shift assay with HAF GST-fusion protein. Increasing amounts (5, 10, and 20 μg) of GST protein (lanes 2-4) or HAF GST-fusion protein (lanes 5-10) were incubated with 100 ng of poly dI-dC (lanes 5-7), or 500 ng of poly dI-dC (lanes 2-4 and 8-10). Lanes 11 and 12 represent binding reactions with 10 μg of GST-fusion protein in the presence of 100- and 500-fold molar excess of the specific competitor EP31. Lane 1 represents the binding reaction with no protein. Each of these reactions was carried out in the presence of 2 μg of BSA as carrier protein. The arrow indicates the shifted complex. F is free probe.
Madhu Gupta et al. Blood 2000;96:
©2000 by American Society of Hematology

3. Super-shift assay with HAF antibody and Hep3B nuclear extract
Super-shift assay with HAF antibody and Hep3B nuclear extract.(A) Western blot showing the specificity of HAF antibody utilized in the supershift assay.
Super-shift assay with HAF antibody and Hep3B nuclear extract.(A) Western blot showing the specificity of HAF antibody utilized in the supershift assay. Arrows indicate the two proteins discussed in the text. (B) Increasing amounts (1, 5, and 10 μL) of anti-HAF serum were incubated with Hep3B nuclear proteins (5 μg) in the presence of the EP17 probe (lanes 2-4). Lane 1 is a control reaction with nuclear proteins alone. Control reactions with preimmune serum in the presence (lane 5) or absence (lane 6) of nuclear extract. Imm. Serum and P.I. Serum refers to immune and preimmune serum respectively, N.E. refers to nuclear extract. S, NS, and SS represent specific, nonspecific, and super-shifted complexes, respectively.
Madhu Gupta et al. Blood 2000;96:
©2000 by American Society of Hematology

4. Functional role of HAF in hypoxia-induced expression of the epo gene
Functional role of HAF in hypoxia-induced expression of the epo gene.(A) Northern analysis of Hep3B clones expressing HAF-antisense RNA. Individual clones (1-8) stably transfected with either HAF cDNA in reverse orientation (lanes 1-7) or the vector alone (...
Functional role of HAF in hypoxia-induced expression of the epo gene.(A) Northern analysis of Hep3B clones expressing HAF-antisense RNA. Individual clones (1-8) stably transfected with either HAF cDNA in reverse orientation (lanes 1-7) or the vector alone (lane 8); lanes 9 and 10 represent controls with non-transfected Hep3B cells and NN10 cells. Total RNA was isolated, size fractionated and hybridized with the HAF cDNA probe. The thin arrow indicates the 2.6-kb HAF transcript. The thick arrow indicates the presence of the 0.7-kb HAF antisense-RNA in clones 2, 4, 5, and 6 but not in wild-type (clone 9) or clones transfected with vector alone (8). The bottom panel shows β-actin mRNA. (B) Effect of HAF-antisense-RNA on hypoxic induction of epo mRNA. Two clones (4 and 5 of panel A) expressing HAF antisense RNA (lanes 3-6), and a control clone (8 of panel A) (lanes 1 and 2) transfected with vector alone were exposed to hypoxia (2% O2) for 48 hours (lanes 2, 4, and 6), or under normal oxygenation (lanes 1, 3, and 5). The probe used was a monkey epo cDNA. The arrow indicates 1.4-kb epo mRNA. (C) Effect of HAF antisense RNA on hypoxia induced expression of VEGF mRNA. The membrane in panel B was stripped of the epo probe and hybridized to human VEGF cDNA. Arrows indicate different transcripts of VEGF. (D) The same membrane was stripped and rehybridized with mouse β-actin cDNA to control for loading differences.
Madhu Gupta et al. Blood 2000;96:
©2000 by American Society of Hematology

5. Gel shift assay with VEGF UTR binding to HAF protein and nuclear proteins of Hep3B cells.(A) Lane 1, probe alone (30 mer); lane 2, GST-HAF + probe; lanes 3, 4, self competition at 100 and 500 molar excess; lanes 5, 6, lack of competition with EP22 at 100 an...
Gel shift assay with VEGF UTR binding to HAF protein and nuclear proteins of Hep3B cells.(A) Lane 1, probe alone (30 mer); lane 2, GST-HAF + probe; lanes 3, 4, self competition at 100 and 500 molar excess; lanes 5, 6, lack of competition with EP22 at 100 and 500 molar excess; lanes 7, 8, competition with EP17 at 100 and 500 molar excess; lane 9, GST only. (B) Lane 1, probe alone (30 mer); lane 2, 3, nuclear proteins (5 and 10 μg) + probe; lanes 4, self-competition at 100 fold molar excess using 10 μg nuclear proteins; lanes 5, 6, GST-HAF (2 and 5 μg) + probe. The bottom of the gel contains the VEGF 30 mer probe (VG30). The arrows indicate the specific complex.
Madhu Gupta et al. Blood 2000;96:
©2000 by American Society of Hematology

6. Northern analysis of HAF expression in different tissues and at different development stages in liver and kidney.The probe used was 700 bp (nt 1-700) of HAF cDNA.
Northern analysis of HAF expression in different tissues and at different development stages in liver and kidney.The probe used was 700 bp (nt 1-700) of HAF cDNA. Total RNA from NN10 cells was used as a positive control. (A) A 2.6-kb single transcript was identified in all the tissues indicated above each lane with the exception of the liver. The bottom panel shows ethidium bromide stained 28 S rRNA. (B) Expression of HAF in liver at indicated development ages. The bottom panel shows ethidium bromide stained 28 S RNA. (C) Quantitative analysis of panel B using densitometer scanning. Scanner units were normalized for 28 S RNA. (D) Expression of HAF in kidneys at different development stages. The arrow indicates 2.6-kb HAF mRNA in upper panel and 28 S rRNA in bottom panel.
Madhu Gupta et al. Blood 2000;96:
©2000 by American Society of Hematology