Effect of the PLCγ inhibitor (PAO) and Src kinase inhibitor (PP2) on leptin-induced TNF-α expression in Raw 264.7 cells.

<p>(A) Raw 264.7 cells were treated with leptin (20 nM) for 5 min. Cells were harvested, and cell extracts were subjected to immunoblotting for PLCγ and Src kinase, respectively. (B) Cells were pretreated with PAO (20 µM) for 30 min and then stimulated with leptin (20 nM) for 5 min. The cell lysates were then analyzed by Western blotting using total Src and p-Src antibodies. (C) Cells were labeled with 2 µCi/ml [³H]-palmitic acid and then pretreated with PAO or PP2 (20 µM) for 30 min before stimulation with leptin (20 nM) for 15 min. PLD activity was determined by estimating the formation of [³H]-PBt in the presence of 1-butanol. Results are the mean ± S.D. of three independent experiments. *p<0.05 vs leptin-treated control (D,E) Cells were pretreated with PAO or PP2 (20 µM) for 30 min and then stimulated with leptin (20 nM) for 30 min. Total RNA was isolated using TRIzol reagent, and mRNA levels were determined by semi-quantitative and real-time RT-PCR with primers for TNF-α or GAPDH. *p<0.05 vs leptin-treated control. (F) Cells in 96-well culture plates were pretreated with PAO or PP2 (20 µM) for 30 min and then stimulated with leptin (20 nM) for 1 h. Results are the mean ± S.E. amounts of TNF-α measured by ELISA for each group of samples. Data are means ± S.E. of eight values. *p<0.05 vs leptin-treated control.