Deciphering the Intracellular Signaling of Erythropoietin in Neuronal Cells - Tissue-Protective Cytokines: Methods and Protocols - page 177

3. Centrifuge the cell lysate/immobilized antibody at 10,000 ´ g
for 30 s.
4. Wash the pellets twice with 500 μ l of lysis buffer, and then with

l of kinase buffer (25 mM Tris-HCl (pH 7.5), 5 mM
b-glycerophosphate, 2 mM dithiothreitol (DTT), 0.1 mM
Na 3 VO 4 , 10 mM MgCl 2 ).
5. Add 50

500 μ

l of
GSK-3 Fusion Protein (Cell Signaling) to the pellets. Incubate
for 30 min at 30°C.
6. Terminate the reaction with 25

μ l of kinase buffer, 1 μ l of 10 mM ATP, and 1 μ

μ l of 4× LDS Sample Buffer

(Life Technologies).
7. Heat the sample to 70°C for 10 min.
8. Load 5-15 l of sample per well on a 10% Bis-Tris SDS-PAGE

gel and transfer to a nitrocellulose membrane. After blocking,
incubate the membrane with Phospho-GSK-3a/b (Ser21/9)
Antibody (1:1,000, Cell Signaling) overnight at 4°C. After
washing, incubate the membrane with HRP-conjugated sec-
ondary antibody (1:2,000) and HRP-conjugated anti-biotin
antibody (1:1,000) to detect biotinylated protein markers.
After washing, incubate the membrane with 10 mL LumiGLO ®
Substrate (Cell Signaling) for 1 min and expose to X-ray film.

μ

1. Treated glass coverslips with a wash in 70% nitric acid under
strong agitation for 2-3 days, followed by a rinse with water
for 4-5 h and a wash in 100% ethanol for 20-30 min. Autoclave
the coverslips on a dry cycle for 15-20 min.
2. Coat tissue culture plates or glass coverslips with poly- L -lysine
(100

3.3.5 Primary Neuronal
Cultures

g/ml in borate buffer) overnight at room temperature.
Then wash the plates three times in water and allow to dry
overnight.
3. Euthanize apregnant Sprague-Dawley rat at embryonic day 17
(E17) with an isoflurane overdose.
4. Sterilize the lower abdomen is with ethanol and open with scis-
sors. Expose the uterus and remove each embryo and place
them in ice-cold Hank's balanced salt solution (HBSS).
5. Remove the heads from each embryo with scissors and place in
fresh ice-cold HBSS. To remove the brains from the skull, cut
up the midline of the skull with scissors, peel away the top lay-
ers of tissue (skin + skull) to expose the cortical hemispheres,
then pinch out the brain from the spinal cord using curved
forceps, and transfer the brain to ice-cold HBSS.
6. Under a dissecting microscope, turn the brain to the dorsal
view. Dissect one hemisphere at a time by cutting the proximal
and distal ends of the cortex with small scissors (about
1 mm).

μ