Insulin Signaling Pathway Activation

Activation of the hIR was assessed by immunoblotting as previously described^{54 (link)}. L6 myoblasts overexpressing IR-A were stimulated with 10 nM insulin or mini-Ins for different times. Cell lysates were subjected to reducing 10 % SDS-PAGE, transferred to nitrocellulose and immunoblotted with the PathScan^® Multiplex Western Cocktail I (Cell Signaling Technology #5301) against pAkt (Ser473), pERK1/2 (Thr202, Tyr204) and mouse anti-β-tubulin (Invitrogen #32–2600). Total Akt and ERK1/2 levels do not change over the time course measured (not shown). The β-tubulin was used as a loading control and pAkt and pERK1/2 were normalized against this control. Quantification of the blots was achieved using the Image Studio Lite quantification software. Activation was expressed as a percentage of the response to insulin at 10 min.

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Xiong X., Menting J.G., Disotuar M.M., Smith N.A., Delaine C., Ghabash G., Agrawal R., Wang X., He X., Fisher S.J., MacRaild C.A., Norton R.S., Gajewiak J., Forbes B.E., Smith B.J., Safavi-Hemami H., Olivera B., Lawrence M.C, & Hung-Chieh Chou D. (2020). A structurally minimized yet fully active insulin based on cone-snail venom insulin principles. Nature structural & molecular biology, 27(7), 615-624.

Publication 2020

PathScan® Multiplex Western Cocktail I mouse anti-β-tubulin

Cell Erk1 Insulin Mouse Myoblasts Nitrocellulose Sds page Tubulin

Corresponding Organization :

Other organizations : University of Utah, Walter and Eliza Hall Institute of Medical Research, La Trobe University, Flinders University, Monash University, University of Copenhagen, University of Melbourne

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Variable analysis

independent variables

Insulin concentration (10 nM)
Mini-Ins treatment

dependent variables

Phosphorylation of Akt (Ser473)
Phosphorylation of ERK1/2 (Thr202, Tyr204)

control variables

L6 myoblasts overexpressing IR-A
Reducing 10% SDS-PAGE
Transferred to nitrocellulose and immunoblotted
PathScan® Multiplex Western Cocktail I antibodies
β-tubulin as loading control
Quantification using Image Studio Lite software

controls

Positive control: 10 min insulin stimulation
Negative control: Not mentioned

Annotations

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