IGF-1 down-regulates Bim expression in the human OPM-2 cells through the p42/44 MAPK and PI-3K pathways. (A) Western blot analysis showing down-regulation of Bim at the protein level in the OPM-2 cells. Cells deprived of serum for 24 hours 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. Variation between experiments was below 10%. (B) IGF-1 inhibits Bim transcription through the PI3-K pathway in the OPM-2 cells. Cells were starved 24 hours, pretreated with LY294002 (1 hour of preincubation; 10μM), and stimulated with IGF-1 for 10 minutes. Next, the levels of p-Akt, tot-Akt, p-FoxO3a, tot-FoxO3a, p-ERK1/2, tot-ERK1/2, and actin were analyzed by Western blot. (C) Activation of the p42/44 MAPK pathway on IGF-1 treatment is delayed in the OPM-2 cells. Cells were deprived of serum for 24 hours before stimulation for the indicated time periods, and the levels of p-ERK1/2 and actin were analyzed by Western blot. (D-E) IGF-1 induces proteasomal degradation of the BimEL isoform by the p42/44 MAPK pathway. (D) OPM-2 cells were deprived of serum for 24 hours 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. (E) Cells were starved for 24 hour, pretreated for 1 hour with 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.