Figure 4.
Figure 4. IL-6 activates Erk5 in myeloma cells through a Ras- and Src-independent route. (A) MM1S cells were infected with pLZR-IRES-GFP or pLZR-RasN17-IRES-GFP, and the expression of Ras was analyzed by Western blot of cell extracts using an anti–H-Ras antibody (iv). The effect of IL-6 on Erk5 (i) and pErk1/2 (ii) is shown, as well as the total Erk1/2 levels (iii). The asterisk indicates a nonspecific band that appears sometimes in the anti-pErk1/2 blots. (B) Quantitative analyses of the levels of Erk5 phosphorylation (□) or pErk1/2 (▪) in MM1S cells infected with pLZR-IRES-GFP or pLZR-RasN17-IRES-GFP, and treated, where indicated, with IL-6 (5 nM, 15 minutes). The results are represented as the mean ± SD of 3 different experiments, and considering as 100% the stimulation obtained with IL-6 in control infected cells. The number (#) sign indicates P < .01. (C) Effect of FPTIII on Erk5 phosphorylation in MM1S cells. Cells were preincubated with FPTIII (10 μM) for 24 hours and then treated with IL-6 for 15 minutes. Erk5, Erk1/2, and pErk1/2 were analyzed as described in A. (D) Quantitative analyses of the effect of FPTIII on Erk5 (□) and Erk1/2 (▪) phosphorylation. The results were quantitated as described for panel B. The number sign (#) indicates P < .01. (E) Effect of the Src inhibitor PP2 on IL-6–induced Erk5 activation. PP2 (20 μM) was added to MM1S cells for the times indicated and then cells were stimulated with IL-6 for 15 minutes. Erk5 was analyzed as described in A. (F) Action of PD98059 on IL-6–induced Erk5 and Erk1/2 phosphorylation. MM1S cells were preincubated with the indicated concentrations of PD98059 for 60 minutes, and then IL-6 added for an additional 15-minute period. Erk5 and pErk1/2 were analyzed by Western blotting as described in A, and the amount of phosphorylation quantitated and graphically represented at the bottom of each panel. (G) Action of IL-6 and PMA on Ras, Mek1, Erk1/2, and Erk5. MM1S cells were treated with IL-6 (5 nM) or PMA (1 μM) for 15 minutes and then lysates prepared for Ras pull-down using GST-RafRBD, pMek, or pErk1/2 analyses on Western blots of cell extracts, or Erk5 analysis on Western blots of anti-Erk5 immunoprecipitates. Results are representative of an experiment that was repeated 3 times.

IL-6 activates Erk5 in myeloma cells through a Ras- and Src-independent route. (A) MM1S cells were infected with pLZR-IRES-GFP or pLZR-RasN17-IRES-GFP, and the expression of Ras was analyzed by Western blot of cell extracts using an anti–H-Ras antibody (iv). The effect of IL-6 on Erk5 (i) and pErk1/2 (ii) is shown, as well as the total Erk1/2 levels (iii). The asterisk indicates a nonspecific band that appears sometimes in the anti-pErk1/2 blots. (B) Quantitative analyses of the levels of Erk5 phosphorylation (□) or pErk1/2 (▪) in MM1S cells infected with pLZR-IRES-GFP or pLZR-RasN17-IRES-GFP, and treated, where indicated, with IL-6 (5 nM, 15 minutes). The results are represented as the mean ± SD of 3 different experiments, and considering as 100% the stimulation obtained with IL-6 in control infected cells. The number (#) sign indicates P < .01. (C) Effect of FPTIII on Erk5 phosphorylation in MM1S cells. Cells were preincubated with FPTIII (10 μM) for 24 hours and then treated with IL-6 for 15 minutes. Erk5, Erk1/2, and pErk1/2 were analyzed as described in A. (D) Quantitative analyses of the effect of FPTIII on Erk5 (□) and Erk1/2 (▪) phosphorylation. The results were quantitated as described for panel B. The number sign (#) indicates P < .01. (E) Effect of the Src inhibitor PP2 on IL-6–induced Erk5 activation. PP2 (20 μM) was added to MM1S cells for the times indicated and then cells were stimulated with IL-6 for 15 minutes. Erk5 was analyzed as described in A. (F) Action of PD98059 on IL-6–induced Erk5 and Erk1/2 phosphorylation. MM1S cells were preincubated with the indicated concentrations of PD98059 for 60 minutes, and then IL-6 added for an additional 15-minute period. Erk5 and pErk1/2 were analyzed by Western blotting as described in A, and the amount of phosphorylation quantitated and graphically represented at the bottom of each panel. (G) Action of IL-6 and PMA on Ras, Mek1, Erk1/2, and Erk5. MM1S cells were treated with IL-6 (5 nM) or PMA (1 μM) for 15 minutes and then lysates prepared for Ras pull-down using GST-RafRBD, pMek, or pErk1/2 analyses on Western blots of cell extracts, or Erk5 analysis on Western blots of anti-Erk5 immunoprecipitates. Results are representative of an experiment that was repeated 3 times.

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