Mouse embryonic fibroblasts (MEFs) were generated from 13.5-day-old embryos obtained from heterozygous PKBa mice intercrosses (Yang et al., 2003). Briefly, after dissection of head and visceral organs for genotyping, embryos were minced and trypsinized for 30 min at 37°C. Embryonic fibroblasts were then plated and maintained in Dulbecco’s Modified Eagle Medium (DMEM) with 10% foetal calf serum (FCS) (Life Technologies), 100 units/ml of penicillin and 100 mg/ml of streptomycin at 37°C in an atmosphere of 5% CO2. All experiments were performed with wild-type and PKBa-/- MEFs between 15-20 passages. To induce adipocyte differentiation, 2-day-postconfluent cells (day 0) were treated with DMEM supplemented with 10% FCS, 8 mg/ml biotin, 4 mg/ml pantothenate, 0.5 mM 3-isobutyl-1-methylxanthine, 1 mM dexamethasone and 10 mg/ml insulin (all from Sigma). Total RNA was extracted from cells using TRIzol (Invitrogen) according to the manufacturer’s instructions.
Label
biotin
Label protocol
Approximately 8 µg of total RNA was processed to produce biotinylated cRNA targets.
Hybridization protocol
standard Affymetrix procedures
Scan protocol
standard Affymetrix procedures
Description
PKBa -/- mouse embryonic fibroblasts (MEFs) were generated from 13.5-day-old embryos
