Figure 1
Figure 1. IGF-1 down-regulates Bim expression in the murine 5T33MMvv cells through the PI-3K and p42/44 MAPK pathways. (A) Microarray analysis of the murine 5T33MMvv cells showing relative down-regulation of Bim mRNA expression after treatment with IGF-1 for 12 hours. The mean value ± SD of 3 independent experiments is given (*P < .001). The Bim gene is represented on the mouse 430A microarrays from the Affymetrix chip by 5 different probe sets. (B) Western blot analysis showing down-regulation of Bim at protein level in the 5T33MMvv cells. Cells deprived of serum for 1 hour were treated with IGF-1 for 24 hours. Equivalent amounts of lysates were immunoblotted with anti-Bim and anti-actin to confirm equal loading. The numbers below refer to the optical density of the BimEL, BimL, and BimS bands after treatment with IGF-1 as measured with the NIH1.62 image program. The variation between experiments was below 10%. (C-D) IGF-1 inhibits Bim transcription through the PI3-K pathway in the 5T33MMvv cells. (C) Western blot analysis showing activation of the PI-3K and p42/44 MAPK pathway after IGF-1 stimulation. Cells were starved 1 hour before IGF-1 treatment for the indicated time periods. The expression of p-ERK1/2, tot-ERK1/2, p-Akt, tot-Akt, and pFoxO3a were analyzed by Western blot. (D) Cells were starved 1 hour, treated with the PI-3K inhibitor LY294002 (1 hour of preincubation; 10μM) or not and stimulated or not with IGF-1 for 10 minutes, and the levels of p-Akt, tot-Akt, p-FoxO3a, and tot-FoxO3a were analyzed by Western blot. To confirm equal loading, levels of actin were determined. (E-F) IGF-1 induces proteasomal degradation of the BimEL isoform by the p42/44 MAPK pathway. (E) Cells were deprived of serum for 1 hour before stimulation for the indicated time periods, and the levels of phosphorylation and expression of BimEL were analyzed by Western blot. Equivalent amounts of lysates were immunoblotted with anti-Bim and anti-actin to confirm equal loading. (F) Cells were starved for 1 hour, pretreated or not for 1 hour with the specific proteasome inhibitor MG132 (1μM) and treated with IGF-1 for the indicated time periods. Next, the levels of Bim were analyzed by Western blot. Equivalent amounts of lysates were immunoblotted with anti-Bim and anti-actin to confirm equal loading. For all Western blot analyses, 1 experiment representative of 3 is shown.

IGF-1 down-regulates Bim expression in the murine 5T33MMvv cells through the PI-3K and p42/44 MAPK pathways. (A) Microarray analysis of the murine 5T33MMvv cells showing relative down-regulation of Bim mRNA expression after treatment with IGF-1 for 12 hours. The mean value ± SD of 3 independent experiments is given (*P < .001). The Bim gene is represented on the mouse 430A microarrays from the Affymetrix chip by 5 different probe sets. (B) Western blot analysis showing down-regulation of Bim at protein level in the 5T33MMvv cells. Cells deprived of serum for 1 hour were treated with IGF-1 for 24 hours. Equivalent amounts of lysates were immunoblotted with anti-Bim and anti-actin to confirm equal loading. The numbers below refer to the optical density of the BimEL, BimL, and BimS bands after treatment with IGF-1 as measured with the NIH1.62 image program. The variation between experiments was below 10%. (C-D) IGF-1 inhibits Bim transcription through the PI3-K pathway in the 5T33MMvv cells. (C) Western blot analysis showing activation of the PI-3K and p42/44 MAPK pathway after IGF-1 stimulation. Cells were starved 1 hour before IGF-1 treatment for the indicated time periods. The expression of p-ERK1/2, tot-ERK1/2, p-Akt, tot-Akt, and pFoxO3a were analyzed by Western blot. (D) Cells were starved 1 hour, treated with the PI-3K inhibitor LY294002 (1 hour of preincubation; 10μM) or not and stimulated or not with IGF-1 for 10 minutes, and the levels of p-Akt, tot-Akt, p-FoxO3a, and tot-FoxO3a were analyzed by Western blot. To confirm equal loading, levels of actin were determined. (E-F) IGF-1 induces proteasomal degradation of the BimEL isoform by the p42/44 MAPK pathway. (E) Cells were deprived of serum for 1 hour before stimulation for the indicated time periods, and the levels of phosphorylation and expression of BimEL were analyzed by Western blot. Equivalent amounts of lysates were immunoblotted with anti-Bim and anti-actin to confirm equal loading. (F) Cells were starved for 1 hour, pretreated or not for 1 hour with the specific proteasome inhibitor MG132 (1μM) and treated with IGF-1 for the indicated time periods. Next, the levels of Bim were analyzed by Western blot. Equivalent amounts of lysates were immunoblotted with anti-Bim and anti-actin to confirm equal loading. For all Western blot analyses, 1 experiment representative of 3 is shown.

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