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. 2006 Apr;26(8):3039–3047. doi: 10.1128/MCB.26.8.3039-3047.2006

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FIG.4. — cAMP inhibits activation of MEKK2 and Raf-1. (A) HeLa cells grown to confluence and cultured in 0.5% FBS for 18 h were treated for 15 min with DMSO or 10 μM forskolin and 50 μM IBMX and then for 5 min with 100 ng/ml EGF. (Top two panels) MEKK2 immune complex kinase assay using His-MEK6KM as the substrate. MEKK2 autophosphorylation (top) and MEKK2 activity towards MEK6KM (second from top) are shown. (Bottom two panels) MEKK2 immunoblot of MEKK2 immune complex kinase assay (third from top) and ERK5 immunoblot of cell lysates (bottom) are shown. (B) NIH 3T3 cells grown to confluence and cultured in 0.5% calf serum for 24 h were treated for 15 min with DMSO or 10 μM forskolin and 50 μM IBMX and then for 5 min with 25 ng/ml FGF. (Top) MEKK2 immune complex kinase assay using His-MEK6KM as the substrate. MEKK2 autophosphorylation and MEKK2 activity towards MEK6KM are shown. (Bottom) MEKK2 immunoblot of MEKK2 immune complex kinase assay. Results shown in panels A and B are representative of at least three independent experiments. (C) HeLa cells grown to confluence and cultured in 0.5% FBS for 18 h were treated for 15 min with DMSO or 10 μM forskolin and 50 μM IBMX and then for 5 min with 100 ng/ml EGF. (Top) Raf-1 immunoblot of Raf-1 immune complex kinase assay. (Second from top) Raf-1 immune complex kinase assay using His-MEK1KM as the substrate. The increase (n-fold) in Raf-1 activity (third panel from top) determined in immune complex kinase assays is the average of results from three independent experiments. Error bars show standard error of the mean. (Bottom) ERK1/2 activity monitored as described for Fig. 1.

FIG.4. — cAMP inhibits activation of MEKK2 and Raf-1. (A) HeLa cells grown to confluence and cultured in 0.5% FBS for 18 h were treated for 15 min with DMSO or 10 μM forskolin and 50 μM IBMX and then for 5 min with 100 ng/ml EGF. (Top two panels) MEKK2 immune complex kinase assay using His-MEK6KM as the substrate. MEKK2 autophosphorylation (top) and MEKK2 activity towards MEK6KM (second from top) are shown. (Bottom two panels) MEKK2 immunoblot of MEKK2 immune complex kinase assay (third from top) and ERK5 immunoblot of cell lysates (bottom) are shown. (B) NIH 3T3 cells grown to confluence and cultured in 0.5% calf serum for 24 h were treated for 15 min with DMSO or 10 μM forskolin and 50 μM IBMX and then for 5 min with 25 ng/ml FGF. (Top) MEKK2 immune complex kinase assay using His-MEK6KM as the substrate. MEKK2 autophosphorylation and MEKK2 activity towards MEK6KM are shown. (Bottom) MEKK2 immunoblot of MEKK2 immune complex kinase assay. Results shown in panels A and B are representative of at least three independent experiments. (C) HeLa cells grown to confluence and cultured in 0.5% FBS for 18 h were treated for 15 min with DMSO or 10 μM forskolin and 50 μM IBMX and then for 5 min with 100 ng/ml EGF. (Top) Raf-1 immunoblot of Raf-1 immune complex kinase assay. (Second from top) Raf-1 immune complex kinase assay using His-MEK1KM as the substrate. The increase (n-fold) in Raf-1 activity (third panel from top) determined in immune complex kinase assays is the average of results from three independent experiments. Error bars show standard error of the mean. (Bottom) ERK1/2 activity monitored as described for Fig. 1.