Induction of signaling was assessed by immunoblotting as described51 (link). L6 myoblasts overexpressing IR-A (240,000 cells/well) were seeded in 6-well plates, grown to confluence (~48 hours), and stimulated with 10 nM human insulin or Vh-Ins-HALQ for different times. Lysates were precipitated with trichloroacetic acid, pH neutralized with 1M Tris pH8.0, separated on 10% SDS-PAGE, transferred to nitrocellulose membrane, and immunoblotted with primary antibodies for 16 h at 4 °C. Antibodies used were phospho-AKT (T308) (New England Biolabs #9275S), phospho p44/42 MAPK (ERK1/2) (T202/Y204) (New England Biolabs #9101S) and mouse anti-β-tubulin (Invitrogen #32–2600). β-tubulin was used as loading control for pAKT and pERK1/2 normalization. Quantitation of blots was performed using Image Studio Lite software. Activation was expressed as a percentage of the response to insulin at 10 min (three independent experiments).
Protocol detail
Insulin Signaling Pathway Activation Assay
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Antibodies
Cells
Erk1
Human
Insulin
Membrane
Mouse
Myoblasts
Nitrocellulose
Sds page
Trichloroacetic acid
Tris
Tubulin
Corresponding Organization : University of Copenhagen
Other organizations : University of Melbourne, Max Planck Institute of Biochemistry, TU Dresden, German Center for Diabetes Research, Heinrich Heine University Düsseldorf, Deutsches Diabetes-Zentrum e.V., University Hospital Carl Gustav Carus, Max Planck Institute of Molecular Cell Biology and Genetics, Paul Langerhans Institute Dresden, Flinders Medical Centre, Flinders University, University of Kentucky
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Variable analysis
independent variables
- Stimulation with 10 nM human insulin
- Stimulation with Vh-Ins-HALQ
- Phosphorylation of AKT (T308)
- Phosphorylation of p44/42 MAPK (ERK1/2) (T202/Y204)
- L6 myoblasts overexpressing IR-A (240,000 cells/well)
- Cells grown to confluence (~48 hours)
- Lysates precipitated with trichloroacetic acid, pH neutralized with 1M Tris pH8.0
- Samples separated on 10% SDS-PAGE, transferred to nitrocellulose membrane
- Immunoblotting with primary antibodies for 16 h at 4 °C
- β-tubulin used as loading control for pAKT and pERK1/2 normalization
- Positive control: Stimulation with 10 nM human insulin
- Negative control: Not explicitly mentioned
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