Immunoblotting Analysis of Cellular Signaling

Proteins were isolated from treated THP-1 cells or cutaneous excised tissues using radioimmunoprecipitation buffer with protease inhibitors (Sigma). Protein concentrations were determined by a Bradford assay (Pierce). The total protein (30 μg) of each sample was subjected to SDS-PAGE and immunoblotting with the desired antibodies against TLR4 (Abcam), STAT3 (Cell Signaling), p-STAT3 (Cell Signaling), AKT (Cell Signaling), p-AKT (Cell Signaling), NF-κB (Cell Signaling), p-P65 (Cell Signaling) and β-actin (Abcam), as previously described [27 (link)].

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Ti D., Hao H., Tong C., Liu J., Dong L., Zheng J., Zhao Y., Liu H., Fu X, & Han W. (2015). LPS-preconditioned mesenchymal stromal cells modify macrophage polarization for resolution of chronic inflammation via exosome-shuttled let-7b. Journal of Translational Medicine, 13, 308.

Publication 2015

protease inhibitors STAT3 p-STAT3 p-AKT NF-κB β-actin

Actin Antibodies Assay Buffer Cutaneous tissues Nf κb Protease inhibitors Protein Radioimmunoprecipitation Sds page Stat3 Thp 1 cells

Corresponding Organization :

Other organizations : Chinese PLA General Hospital, Hainan Branch of People's Liberation Army General Hospital

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Variable analysis

independent variables

Treatment (treated THP-1 cells or cutaneous excised tissues)

dependent variables

Protein levels of TLR4
Protein levels of STAT3
Phosphorylation levels of STAT3 (p-STAT3)
Protein levels of AKT
Phosphorylation levels of AKT (p-AKT)
Protein levels of NF-κB
Phosphorylation levels of p65 (p-P65)
Protein levels of β-actin

control variables

Total protein (30 μg) of each sample
Protease inhibitors (Sigma) used in protein isolation

controls

Positive control: Not specified
Negative control: Not specified

Annotations

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