Tyrosine phosphorylation of Felic.

HEK293 cells were transfected for 30 hours with pCMVFLAG-Felic (Felic-FLAG +) or empty vector (−). Cells were then grown overnight in RPMI depleted of serum and 2% bovine serum albumin, then stimulated with 10% FCS or 100 ng/mL insulin for 15 minutes at 37°C. Lysates were prepared and immunoprecipitated (IP) with FLAG-antibody, and blotted with antiphosphotyrosine mAb 4G10 (Upstate Biotechnology, Lake Placid, NY). Lower panel shows comparable levels of immunoprecipitated Felic. WCLs show expression of Felic.