Western Blotting for Protein Expression Analysis

Western blotting assay was carried out as described^{45 (link),46 (link)}. Briefly, tissue or cell lysates were prepared in Laemmli Sample Buffer. Equal amounts of total proteins were subjected SDS-PAGE and transferred to PVDF membranes, which were blocked and incubated overnight with the primary antibodies against Akt (Santa Cruz Biotechnology), p-Akt (Santa Cruz Biotechnology), Col1a (Abcam), fibronectin (Abcam), α-Sma (Abcam), or Gapdh (Abcam). After being washed, the membranes were incubated with respective secondary antibodies conjugated with horseradish peroxidase (HRP). Immune-reactive signals were visualized by the Enhanced Chemiluminescence (ECL) kit (Millipore, America) on Syngene PXi6 Access imaging system (Frederick, MD). The band intensities were quantified using Image Pro Plus 6.0.

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Yu X., Xia Y., Zeng L., Zhang X., Chen L., Yan S., Zhang R., Zhao C., Zeng Z., Shu Y., Huang S., Lei J., Yuan C., Zhang L., Feng Y., Liu W., Huang B., Zhang B., Luo W., Wang X., Zhang H., Haydon R.C., Luu H.H., He T.C, & Gan H. (2018). A blockade of PI3Kγ signaling effectively mitigates angiotensin II-induced renal injury and fibrosis in a mouse model. Scientific Reports, 8, 10988.

Publication 2018

p-Akt Col1a fibronectin α-Sma Gapdh Enhanced Chemiluminescence (ECL) kit PXi6 Access imaging system

Antibodies Cell Chemiluminescence Fibronectin Gapdh Horseradish peroxidase Laemmli buffer Membranes Proteins Pvdf Sds page Tissue Western blotting assay

Corresponding Organization :

Other organizations : First Affiliated Hospital of Chongqing Medical University, Chongqing Medical University, University of Chicago Medical Center

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Levels of Akt
Levels of phosphorylated Akt (p-Akt)
Levels of Collagen 1a (Col1a)
Levels of Fibronectin
Levels of Alpha-Smooth Muscle Actin (α-Sma)
Levels of Glyceraldehyde 3-phosphate dehydrogenase (Gapdh)

control variables

Equal amounts of total proteins were subjected to SDS-PAGE

controls

Gapdh was used as a loading control

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