Insulin and IGF-I Signaling Pathway Analysis

Following confluency, pOBs were cultured for 24 hours in serum-free medium containing 0.1% BSA. Cells were then either lyzed immediately or stimulated with insulin (10 nM; Sigma) or IGF-I (10 nM; Sigma) for 10 minutes before lysis. Cells were lysed in RIPA buffer (Invitrogen) containing ‘phosphatase inhibitor cocktail 2’ (Sigma) and ‘complete’ protease inhibitor cocktail (Roche) according to the manufacturers’ instructions. Immunoblotting was conducted with specific antibodies against phospho-Akt^Ser473, total Akt, phospho-GSK3β^Ser9, total GSK3β, phospho-Erk1/2^{Thr202/Tyr204} and total Erk1/2 (Cell Signaling, Boston, MA, USA). Immunoblotting was conducted as previously described (Zhu et al., 2011 (link); Zhu et al., 2013 (link)) and visualized using the enhanced chemiluminescence (ECL) Western Blotting Detection System (GE Healthcare, Chalfont St Giles, UK).