Quantitative Western Blot Analysis of Liver Proteins

Frozen liver tissue from mice and human samples were homogenized in RIPA lysis buffer (Barnes et al., 2013 (link)) with protease and phosphatase inhibitors added and protein concentrations were assayed using the DC Protein Assay (BioRad, Hercules, CA). Liver lysates were then separated on 12% polyacrylamide gels and used for western blot analysis with antibodies against CHOP (#5554), phospho^Ser51 eIF2α (#3597), total eIF2α (#9722) (Cell Signaling, Danvers, MA); MIF (#TP234) (Torrey Pines Biolabs, Inc, Secaucus, NJ), PDI (#610946), GRP78 (#610978) (BD Biosciences, San Jose, CA). HSC70 (sc-7298) (Santa Cruz Biotechnology, Dallas, TX), β-actin (#4967), (Cell Signaling) and GAPDH (MAB374) (Millipore Sigma, St. Louis, MO) were used as loading controls. Signal intensities were quantified using Eastman Kodak Co. Image Station 4000R.

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Poulsen K., McMullen M., Huang E., Kibler C., Sheehan M., Leng L., Bucala R, & Nagy L. (2019). Novel Role of Macrophage Migration Inhibitory Factor in Upstream Control of the Unfolded Protein Response after Ethanol Feeding in Mice. Alcoholism, clinical and experimental research, 43(7), 1439-1451.

Publication 2019

DC Protein Assay phosphoSer51 eIF2α total eIF2α GRP78 HSC70 (sc-7298) β-actin GAPDH (MAB374) Image Station 4000R

Actin Antibodies Assay Buffer Chop Frozen Gapdh Grp78 Human Inhibitors Liver Mice Phosphatase Pines Polyacrylamide gels Protease Protein Ripa Tissue Western blot analysis

Corresponding Organization : Cleveland Clinic

Other organizations : Case Western Reserve University, Yale University

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Variable analysis

independent variables

Tissue source (mouse vs. human)

dependent variables

Protein levels of CHOP, phospho-eIF2α, total eIF2α, MIF, PDI, GRP78

control variables

Protein concentrations assayed using the DC Protein Assay
Proteins separated on 12% polyacrylamide gels
Loading controls: HSC70, β-actin, GAPDH

controls

Positive controls: Not explicitly mentioned
Negative controls: Not explicitly mentioned

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