Western Blot Analysis of Cellular Proteins

Cells were lysed with buffer containing 1% NP-40 and proteinase inhibitor cocktail (Thermo Scientific, Rockford, IL). Protein concentrations were determined by Bradford assay (Bio-Rad) and equalized before loading. Cellular protein from each sample was applied to 8% to 12% SDS-PAGE gels and probed with specific antibodies including EEA1, Rab7, RIP1 (Cell Signaling Technology, Danvers, MA), AnxA2, p-IκBα, p-IKKα, p-ERK, p-p38, p-JNK, p-NFκB p65, p-NFκB p50, TNFα, IL-6, IL-1β, and β-actin (Santa Cruz Biotechnology). Blots were developed with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology) and chemiluminescent substrate on Fuji X-ray films52 (link).

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Zhang S., Yu M., Guo Q., Li R., Li G., Tan S., Li X., Wei Y, & Wu M. (2015). Annexin A2 binds to endosomes and negatively regulates TLR4-triggered inflammatory responses via the TRAM-TRIF pathway. Scientific Reports, 5, 15859.

Publication 2015

proteinase inhibitor cocktail Bradford assay AnxA2 p-IκBα p-IKKα p-ERK p-JNK p-NFκB p65 p-NFκB p50 TNFα IL-6 IL-1β β-actin horseradish peroxidase-conjugated secondary antibodies

Actin Antibodies Anxa2 Assay Buffer Cellular Gels Horseradish peroxidase Ikkα Il 1β Iκbα Nfκb p50 Nfκb p65 Np 40 Protein Proteinase inhibitor Sds page Tnfα X ray

Corresponding Organization :

Other organizations : University of North Dakota, Sichuan University

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Variable analysis

independent variables

Cell lysis buffer containing 1% NP-40 and proteinase inhibitor cocktail

dependent variables

Protein concentrations (measured by Bradford assay)
Levels of proteins (EEA1, Rab7, RIP1, AnxA2, p-IκBα, p-IKKα, p-ERK, p-p38, p-JNK, p-NFκB p65, p-NFκB p50, TNFα, IL-6, IL-1β, β-actin)

control variables

Protein loading (equalized before loading)

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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