Quantitative Western Blot Analysis of xCT

Tissue samples were homogenized using a total protein extraction kit (BioChain) following the manufacturer’s instructions. Protein concentration in lysates was determined by bicinchoninic acid (BCA) protein assay (Pierce). Rabbit anti-xCT antibody (1:2000 dilution, Novus NB300-318) was used according to a standard western blotting protocol. After enhanced chemiluminescence (ECL) exposure, blots were washed and re-probed with rabbit anti-actin antibody (Cell Signaling Technology 4967S; 1:5000) as a loading control. To verify efficient extraction of xCT from the membrane during tissue lysis, an anti-Na,K-ATPase antibody (Cell Signaling Technology 3010S; 1:5000) was used as an additional control for western blotting [16 (link)]. Blots were scanned and signal was quantified using ImageJ (National Institutes of Health).

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Hoehne A., James M.L., Alam I.S., Ronald J.A., Schneider B., D’Souza A., Witney T.H., Andrews L.E., Cropper H.C., Behera D., Gowrishankar G., Ding Z., Wyss-Coray T., Chin F.T., Biswal S, & Gambhir S.S. (2018). [18F]FSPG-PET reveals increased cystine/glutamate antiporter (xc-) activity in a mouse model of multiple sclerosis. Journal of Neuroinflammation, 15, 55.

Publication 2018

total protein extraction kit NB300-318

Actin Anti antibody Antibody Assay Atpase Bicinchoninic acid Chemiluminescence Dilution Membrane tissue Novus Protein Rabbit Tissue

Corresponding Organization : Stanford University

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Variable analysis

independent variables

Tissue samples

dependent variables

XCT protein expression levels
Actin protein expression levels (as a loading control)

control variables

Tissue lysis conditions (using a total protein extraction kit)
Western blotting protocol (standard procedure)
Antibody dilutions (1:2000 for anti-xCT, 1:5000 for anti-actin and anti-Na,K-ATPase)
Signal quantification method (ImageJ)

controls

Positive control: Actin (housekeeping gene) for protein loading
Negative control: Na,K-ATPase (membrane protein) to verify efficient extraction of xCT from the membrane during tissue lysis

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