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. 2006 Oct 18;103(44):16568–16573. doi: 10.1073/pnas.0607822103

Fig. 2.

Fig. 2.

Fisetin activates ERK1 (p44), ERK2 (p42), and CREB in rat hippocampal slices. (A) Hippocampal slices in ACSF were treated with 1 μM fisetin for 5–20 min, and then equal amounts of protein were analyzed by SDS/PAGE and immunoblotting with antibodies to phospho-ERK and phospho-CREB along with antibodies to the unphosphorylated forms of the proteins, demonstrating no changes in overall protein levels. Similar results were obtained in two independent experiments. (B) Hippocampal slices were pretreated for 30 min with either 50 μM PD98059 (PD) or 10 μM U0126 (U) before the addition of 1 μM fisetin for 5 min. Samples were analyzed as in A. (C) The average phosphoprotein signal from the blots in A, quantified by densitometry and normalized to total protein, was plotted ±SD. The asterisks indicate a significant difference from the control (P < 0.005). (p42: 5 min, 1.68 ± 0.06; 10 min, 1.31 ± 0.05; 20 min, 1.42 ± 0.03; p44: 5 min, 1.98 ± 0.16; 10 min, 1.48 ± 0.02; 20 min, 1.30 ± 0.09; CREB: 5 min, 2.87 ± 0.56; 10 min, 2.72 ± 0.12; 20 min, 2.55 ± 0.39.) (D) The average phosphoprotein signal from the blots in B, quantified by densitometry and normalized to total protein, was plotted ±SD. ∗, significant difference from the control (P < 0.0005).#, significant difference from fisetin alone (P < 0.0001). (p42: PD98059, 0.83 ± 0.08; U0126, 0.30 ± 0.05; 5 min fisetin, 1.68 ± 0.06; fisetin + PD98059, 0.72 ± 0.09; fisetin + U0126, 0.54 ± 0.06; p44: PD98059, 0.75 ± 0.05; U0126, 0.13 ± 0.06; 5 min fisetin, 1.98 ± 0.16; fisetin + PD98059, 0.70 ± 0.10; fisetin + U0126, 0.29 ± 0.05; CREB: PD98059, 0.64 ± 0.11; U0126, 0.71 ± 0.31; 5 min fisetin, 2.87 ± 0.56; fisetin + PD98059, 0.55 ± 0.05; fisetin + U0126, 0.20 ± 0.03.) Similar results were obtained in two independent experiments.