Biology Reference
In-Depth Information
responds to hypoxia with increased expression, albeit response in
the brain is magnitudes smaller than in the kidney (
29
). Targeted
deletion of the
HIF1
in astrocytes resulted in over
50% reduction of hypoxia-dependent upregulation of EPO pro-
duction in the brain (
29
). However, this did not cause systemic
anemia, further underlining the distinct non-hematopoietic func-
tion of EPO in the CNS.
α
and
HIF2
α
2
Materials
1. TRIzol
®
(Life Technologies).
2. High Capacity cDNA Reverse Transcription Kit (4368814,
Life Technologies).
3. Primers: primers: EPO: 5 ¢-TCTGCGACAGTCGAGTTCT-3¢
(sense) and 5¢-GTATCCACTGTGAGTGTTCG-3¢ (anti-
sense), located in exon 2 and exon 5 of the murine
EPO
gene
, and 5¢-GGACACCTACTTGGTATTGG-3¢ (sense) and
5¢-GACGTTGTAGGCTGGAGTCC-3¢ (antisense), located
in exon 8 and the 3¢ untranslated region of the murine
EPO
-
R gene
.
4. SYBR Green PCR Master Mix (4367659, Life Technologies).
2.1 Detection of EPO
and EPO-Receptor
mRNA in the Brain
Tissue
1. Cell lysis buffer (50 mM Tris-Cl buffer at pH 8.0, 150 mM
NaCl, 100 mg/ml phenylmethyl sulphonyl fluoride, 1 mg/ml
aprotinin, 1% Triton X-100).
2. BCA-Protein assay kit (Thermo Scientific, # 23225).
3. 4× LDS sample buffer (NuPAGE
®
LDS Sample Buffer,
Invitrogen, NP0007).
4. 10% Bis-Tris SDS-PAGE gels (Invitrogen, # NP0301BOX)
and Nitrocellulose Membrane (Invitrogen, #LC2009).
5. ChemiBlocker (Millipore, #2170).
6. Antibodies raised against EPO-R (R&D Systems), p-JAK2
(Santa Cruz), JAK2 (Santa Cruz), p-STAT-5 (Cell Signaling)
or STAT-5 (Cell Signaling).
7. Horseradish
2.2 Detection
of EPO-Receptor
Protein in the Brain
Tissue ( see Note 1 )
peroxidase
(HRP)
(1:20,000,
Vector
Laboratories).
8. LumiGLO
®
Substrate (Cell Signaling, #7003).
9. X-Ray films.
1. Protein A agarose beads (Cell signaling, #9863).
2. 4× LDS sample buffer (NuPAGE
®
LDS Sample Buffer,
Invitrogen, NP0007).
2.3
Immunoprecipi-
tation
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