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. 2009 Dec 3;59(3):600–610. doi: 10.2337/db09-0897

FIG. 7.

FIG. 7.

Effect of ceramide on insulin-induced phosphorylation of PKB/Akt in control human fibroblast versus caveolin-1–overexpressing human fibroblast. A: Control human fibroblast and caveolin-1–overexpressing human fibroblast lysates were immunoblotted with antibodies against either PKCζ, PP2A, native PKB/Akt, GFP, or caveolin-1. B: Human fibroblasts were treated with 500 μmol/l OKA for 30 min and 100 nmol/l insulin for the last 10 min before being lysed. Cell lysates were immunoblotted with antibodies against either native PKB/Akt or Ser473 PKB/Akt. C: Control human fibroblast and caveolin-1–overexpressing human fibroblast were preincubated with 100 μmol/l C2-ceramide for 2 h, followed by 500 μmol/l OKA for the last 30 min. Then, 100 nmol/l insulin was added to the cells for 10 min before being lysed. Cell lysates were immunoblotted with antibodies against native PKB/Akt, Ser473 PKB/Akt, Ser21/9 GSK3α/β, PKCζ, and GFP. Scanning densitometry was performed to quantify changes in Ser473 PKB/Akt abundance in cell lysates. Bars represent mean ± SEM. *Significant change, P < 0.05 relative to the untreated control. Blots shown represent three to four separate experiments.