Deciphering the Intracellular Signaling of Erythropoietin in Neuronal Cells - Tissue-Protective Cytokines: Methods and Protocols - page 175

3 Methods

3.1 Detection of EPO
and EPO-Receptor
mRNA in the Brain
Tissue

1. Isolate total RNA with TRIzol ® (Life Technologies).
2. Total RNA (1 g) is reverse-transcribed with the commercially

available High Capacity cDNA Reverse Transcription Kit.
3. Use cDNA as a template for PCR amplification of either EPO
or EPO-R with the following primers: 5¢-TCTGCGA-
CAGTCGAGTTCT-3¢ (sense) and 5¢-GTATCCACTGTGAG-
TGTTCG-3¢ (antisense), located in exon 2 and exon 5 of the
murine EPO gene, and 5'-GGACACCTACTTGGTATTGG-3¢
(sense) and 5¢-GACGTTGTAGGCTGGAGTCC-3¢ (anti-
sense), located in exon 8 and the 3¢ untranslated region of the
murine EPO-R gene.
4. Real time PCR is performed using a commercially available
SYBR Green PCR Master Mix. For each sample, a 20

μ

μ l reac-

l of SYBR Green PCR
Master Mix, 200 nM of the reverse primer, 200 nM of the for-
ward primer and 10-100 ng of cDNA template.
5. Program a thermal cycler to hold for 10 min at 95°C, followed
by 40 cycles of 95°C for 15 s and 60°C for 60 s. Real time PCR
products will be 395 bp for EPO and 452 bp for EPO-R.

tion prepare a mixture containing 10 μ

3.2 Detection of EPO
and EPO-Receptor
Protein in the Brain
Tissue

1. Load equivalent amounts of protein (15-30 g) on 10% Bis-

Tris SDS-PAGE gels under reducing conditions at (80 V, 3 h,
RT).
2. Transfer separated proteins onto nitrocellulose membranes.
After nonspecific binding is blocked with ChemiBlocker for
1 h, the blots are subsequently incubated overnight at 4°C
with EPO-R (1:200), p-JAK2 (1:200), JAK2 (1:200),
p-STAT-5 (1:1,000), or STAT-5 (1:1,000). After washing,
membranes are incubated with secondary antibodies conju-
gated to horseradish peroxidase (HRP) (1:20,000) for 1 h and
developed with an enhanced chemiluminescence kit and
exposed to X-ray film.

μ

3.2.1 Western Blotting

3.3 Methods

To detect JAK2 and STAT-5 signaling in neurons, primary cere-
brocortical cultures are lysed in ice-cold cell lysis buffer (50 mM
Tris-Cl buffer at pH 8.0, 150 mM NaCl, 100 mg/ml phenylm-
ethyl sulphonyl fluoride, 1 mg/ml aprotinin, 1% Triton X-100).
The lysates are vortexed for 15 s and centrifuged at 10,000 ´ g for
20 m. The supernatants are used to determine protein concentra-
tions using a BCA-Protein assay kit (Pierce).

3.3.1 Lysates

Equivalent amounts of protein (15-30 g) is resolved on 10% Bis-

Tris SDS-PAGE gels under reducing conditions, and transferred
onto nitrocellulose membranes. After nonspecific binding is

μ

3.3.2 Western Blotting