Journal:

Article Title: Multiple signalling pathways involved in β 2 -adrenoceptor-mediated glucose uptake in rat skeletal muscle cells

doi: 10.1038/sj.bjp.0706626

Figure Lengend Snippet: In (a) [3H]-2-DG uptake (GU) in response to insulin (1 μM) or zinterol (1 μM) was significantly inhibited by the presence of PI3K inhibitor LY294002 (1 μM) (Student's t-test, P=0.01 and P<0.001, n=4–5), whereas the response to 8Br-cAMP (1 mM) was not affected by LY294002 (P=0.9, n=5). In (b) Western blot analysis showed that phosphorylation of Akt at Ser473 occurred in response to insulin (10 μM; 5 min stimulation) but not zinterol (100 nM; 3 h stimulation). Total Akt was detected in all samples. The labels are: basal (1), zinterol (2) and insulin (3). Blots from three separate experiments are shown.

Article Snippet: Samples were harvested in prewarmed (65°C) sodium dodecyl sulfate sample buffer containing 50 m M dithiothreitol, sonicated, boiled for 3 min, and separated on 5% acrylamide gel (or 10% for Akt) for 2 h at 100 V. The proteins were transferred on to Hybond-C extra nitrocellulose (Amersham Biosciences UK Ltd, U.K.) or PVDF membrane (Millipore, Australia), and probed with primary antibodies: phosphor-acetyl-CoA carboxylase (Ser79), (dissolved 1 : 1000 in 5% BSA), phospho-Akt (Ser473) and total Akt (1 : 2000 in 5% BSA) (Cell Signalling Technology, Inc., Danvers, MA, U.S.A.).

Techniques: Western Blot