Figure 2

Effect of celastrol on phorbol ester (PMA) induced phosphorylation of key members of the NFκB signaling pathway. HEK293 cells overexpressing APPsw were treated for 15 minutes with PMA in the presence of 0 μM and 5 μM of celastrol. A) Western blots reveal that celastrol did not inhibit Raf-1 phosphorylation, MEK1/2 phosphorylation or p44/42 MAPK phosphorylation induced by PMA but prevented IKBα phosphorylation induced by PMA suggesting that celastrol is inhibiting IKK activity. B) Histogram representing the quantification of Raf-1 phosphorylation/actin, MEK1/2 phosphorylation/actin, phospho p44/42 MAPK/actin and IKBα phosphorylation/actin chemoluminescent signal. ANOVA shows a significant main effect of PMA on the phosphorylation of these different proteins (P < 0.05). Post-hoc comparisons reveal significant increase in phosphorylation after PMA treatment for Raf-1 (P < 0.04), MEK1/2 (P < 0.01), p44/42 MAPK (P < 0.001) and IKBα (P < 0.001) compared to the control conditions. Celastrol does not appear to prevent PMA induced Raf-1 phosphorylation (P = 0.619) but leads to a significant stimulation of MEK1/2 and p44/42 MAPK compared to PMA treatment alone (P < 0.02) and significantly inhibited IKBα phosphorylation compared to PMA treatment alone (P < 0.001).