Protein Extraction and Western Blot Analysis

Protein extractions were performed as previously described (18 (link),23 ). Primary antibodies phospho-Akt (Ser473) (catalog no. 4060; Cell Signaling Technology) and total Akt (catalog no. 2920; Cell Signaling Technology), GALE (catalog no. ab155997; Abcam), tubulin (catalog no. sc-53030; Santa Cruz Biotechnology), O-linked glycosylation (catalog no. ab2739; Abcam), and actin (catalog no. A4700; Sigma-Aldrich) were used at 1:1,000 dilutions and detected using a secondary immunoglobulin G labeled with infrared dyes emitting at 680 nm (catalog no. 926-68070 and 926-68076; LI-COR Bioscience) or 800 nm (catalog no. 926-32211; LI-COR Bioscience) (both at 1:10,000 dilutions) and then visualized on a LI-COR Odyssey infrared scanner (LI-COR Bioscience). The scanned data were analyzed using Odyssey version 3.0 software (LI-COR Bioscience).

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Zhu Y., Zhao S., Deng Y., Gordillo R., Ghaben A.L., Shao M., Zhang F., Xu P., Li Y., Cao H., Zagnitko O., Scott D.A., Gupta R.K., Xing C., Zhang B.B., Lin H.V, & Scherer P.E. (2017). Hepatic GALE Regulates Whole-Body Glucose Homeostasis by Modulating Tff3 Expression. Diabetes, 66(11), 2789-2799.

Publication 2017

phospho-Akt (Ser473) total Akt LI-COR Odyssey infrared scanner Odyssey version 3.0 software

Actin Antibodies Dilutions Dyes Gale Glycosylation Immunoglobulin g Protein Tubulin

Corresponding Organization : Eli Lilly (United States)

Other organizations : Chinese Academy of Sciences, Shanghai Institutes for Biological Sciences, Discovery Institute, Sanford Burnham Prebys Medical Discovery Institute

Top 5 similar protocols

Variable analysis

independent variables

Protein extractions

dependent variables

Phospho-Akt (Ser473) levels
Total Akt levels
GALE levels
Tubulin levels
O-linked glycosylation levels
Actin levels

control variables

Primary antibodies used at 1:1,000 dilutions
Secondary immunoglobulin G labeled with infrared dyes used at 1:10,000 dilutions
Visualization using LI-COR Odyssey infrared scanner
Data analysis using Odyssey version 3.0 software

controls

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

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