Western Blot Analysis of Spinal Cord

Nuclear and cytosolic fractions of the spinal cords of mice were prepared at six time points. Membrane and cytosolic fractions of cultured astrocytes were also prepared by the same procedure. A total of 20 μg protein lysates was then resolved by 10% SDS-PAGE, transferred to a PVDF membrane, and probed with rabbit monoclonal antibodies against SGK-1 (1:1,000; ab32374, Abcam, Cambridge, UK), GCR (1:1,000; sc1002, Santa Cruz Biotechnology, Santa Cruz, CA), Pannexin-1 (1:1,000; ab124131, Abcam), Actin (1:1,000; sc1616-HRP, Santa Cruz Biotechnology) or POL2 (1:1,000; sc900, Santa Cruz Biotechnology). The specificities of antibodies for their target proteins have been validated in previous studies58 (link)59 (link)60 (link). Specific antigen–antibody complexes were visualized using horseradish peroxidase-conjugated secondary antibodies and a chemiluminescence reagent (Nacalai Tesque, Kyoto, Japan).

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Koyanagi S., Kusunose N., Taniguchi M., Akamine T., Kanado Y., Ozono Y., Masuda T., Kohro Y., Matsunaga N., Tsuda M., Salter M.W., Inoue K, & Ohdo S. (2016). Glucocorticoid regulation of ATP release from spinal astrocytes underlies diurnal exacerbation of neuropathic mechanical allodynia. Nature Communications, 7, 13102.

Publication 2016

ab32374 sc1002 ab124131 sc1616-HRP

A protein Actin Antibodies Antibodies specificities Antigen antibody complexes Astrocytes Chemiluminescence Cytosolic Horseradish peroxidase Membrane Mice Monoclonal antibodies Proteins Pvdf Rabbit Sds page Spinal cords

Corresponding Organization :

Other organizations : Kyushu University, Hospital for Sick Children

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Variable analysis

independent variables

Time points

dependent variables

Protein expression levels of SGK-1, GCR, Pannexin-1, Actin, and POL2

control variables

Protein lysate amount (20 μg)
SDS-PAGE (10%)
Antibody concentrations (1:1,000)
Membrane transfer method (PVDF)
Visualization method (Horseradish peroxidase-conjugated secondary antibodies and chemiluminescence reagent)

positive controls

Validation of antibody specificities for target proteins in previous studies

negative controls

None mentioned

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