Immobilon fl pvdf transfer membrane

To measure the Akt activation
(phosphorylation), L6 myoblasts were differentiated into myotubes
for 7 days, serum starved O/N, and treated with 0–50 μM
sulfonanilides for 15 min after which 10 nM insulin (Actrapid, Novo
Nordisk, Bagsvaerd, Denmark) was added for an additional 15 min. The
cells were lysed in NP-40 buffer (1% nonidet P-40, 20 mM Tris–HCl
pH 7.4, 150 mM NaCl) supplemented with phosphatase inhibitors (50
mM NaF and 1 mM Na₃VO₄) and protease inhibitor
cocktail (cOmpleteProtease Inhibitor Coctail tablets, Roche Diagnostics
GmbH, Mannheim, Germany) and then immunoblotted as in Tolvanen et
al.^{32 (link)} Briefly, samples were separated in
8% polyacrylamide gels and transferred onto PVDF membrane (Immobilon-FL
PVDF transfer membrane, Merck Millipore Ltd., Tullagreen, Ireland).
The membrane was blocked using Odyssey blocking buffer (LI-COR, Lincoln,
NE, USA) and incubated with primary antibodies against pAkt (phospho-Akt,
Ser473, #9271, Cell Signaling Technology, Danvers, MA, USA), Akt (pan-Akt,
#281046, R&D Systems, Minneapolis, MN, USA), and actin (A3853,
Sigma-Aldrich) followed by secondary antibodies (IRDye 800CW Donkey
anti-Rabbit and IRDye 680RD Donkey anti-Mouse, both from LI-COR, Lincoln,
NE, USA). Three individual assays were performed (n = 3 for each condition).