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. Author manuscript; available in PMC: 2008 Jul 1.

Published in final edited form as: Cell Signal. 2007 Feb 8;19(7):1542–1553. doi: 10.1016/j.cellsig.2007.01.028

Fig. 6 — (A) Serum-starved A2780 cells were pretreated with PKC inhibitor bisindolylmaleimide (BIS) for 30 min, followed by incubation with 200 ng/ml IGF-I for 6 h. Total PKC activity was measured using myelin basic protein as a substrate as described in Experimental Procedures. (B) The serum-starved cells were pretreated with BIS and treated with 200 ng/ml IGF-I for 6 h. COX-2 and β–actin protein levels were analyzed by immunoblotting. The lower panel indicated the mean ± SD of the relative densitometry data of COX-2 protein levels from duplicate experiments. (C) The cells were treated as above. COX-2 and GAPDH mRNA levels in the cells were analyzed by RT-PCR. The lower panel indicated the mean ± SD of the relative densitometry data of COX-2 mRNA levels from duplicate experiments. (D and E) The cells were transfected with phPHES2 (−1432/+59) and Luc-3′UTR reporter constructs, respectively. The cells were cultured overnight after the transfection, then switched to serum-free medium in the absence or presence of BIS and 200 ng/ml IGF-I for 16 h. Relative luciferase activity was analyzed in the cells. Data represent mean ± SD from five independent experiments. *indicates that the value is significantly different when compared to that of the control (p<0.05); #indicates that the value is significantly different when compared to that of IGF-I treatment alone (p<0.05). (F) The serum-starved cells were pretreated with BIS, followed by incubation with 200 ng/ml IGF-I for 4 h. Specific protein levels were analyzed by immunoblotting using antibodies against phospho-Erk1/2 (p-Erk1/2, Thr202/Tyr204), and total Erk1/2, respectively. The lower panel indicated the mean ± SD of the relative p-Erk1/2 levels from duplicate experiments.

Fig. 6 — (A) Serum-starved A2780 cells were pretreated with PKC inhibitor bisindolylmaleimide (BIS) for 30 min, followed by incubation with 200 ng/ml IGF-I for 6 h. Total PKC activity was measured using myelin basic protein as a substrate as described in Experimental Procedures. (B) The serum-starved cells were pretreated with BIS and treated with 200 ng/ml IGF-I for 6 h. COX-2 and β–actin protein levels were analyzed by immunoblotting. The lower panel indicated the mean ± SD of the relative densitometry data of COX-2 protein levels from duplicate experiments. (C) The cells were treated as above. COX-2 and GAPDH mRNA levels in the cells were analyzed by RT-PCR. The lower panel indicated the mean ± SD of the relative densitometry data of COX-2 mRNA levels from duplicate experiments. (D and E) The cells were transfected with phPHES2 (−1432/+59) and Luc-3′UTR reporter constructs, respectively. The cells were cultured overnight after the transfection, then switched to serum-free medium in the absence or presence of BIS and 200 ng/ml IGF-I for 16 h. Relative luciferase activity was analyzed in the cells. Data represent mean ± SD from five independent experiments. *indicates that the value is significantly different when compared to that of the control (p<0.05); #indicates that the value is significantly different when compared to that of IGF-I treatment alone (p<0.05). (F) The serum-starved cells were pretreated with BIS, followed by incubation with 200 ng/ml IGF-I for 4 h. Specific protein levels were analyzed by immunoblotting using antibodies against phospho-Erk1/2 (p-Erk1/2, Thr202/Tyr204), and total Erk1/2, respectively. The lower panel indicated the mean ± SD of the relative p-Erk1/2 levels from duplicate experiments.