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. 2009 Nov 17;285(4):2397–2414. doi: 10.1074/jbc.M109.064295

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© 2010 by The American Society for Biochemistry and Molecular Biology, Inc.

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FIGURE 5. — PDK1-mediated inhibition of ASK1 activity is dependent on the modulation of the association between ASK1 and its regulators, 14-3-3 and MKK3, and ASK1 phosphorylation. A and B, modulation of the endogenous association of ASK1 with 14-3-3 protein and MKK3. HEK293 cells were transfected with the indicated combinations of FLAG-14-3-3, FLAG-ASK1, HA-MKK3, HA-ASK1, Myc-tagged PDK1 (WT and KD), GST-PDK1, and PDK1-specific siRNA. Cell lysates were then subjected to immunoprecipitation with the antibodies indicated, and the resulting immunoprecipitates were analyzed by immunoblot analysis using the appropriate antibodies to determine the association of 14-3-3 protein (A) and MKK3 (B) with ASK1. The knockdown effect of endogenous PDK1 on the association between endogenous ASK1 and 14-3-3 protein or MKK3 was determined by immunoblot analysis with the antibodies indicated using HEK293 cells transiently transfected with PDK1-specific siRNA 1 (A and B, right panels). C, HEK293 cells were transiently transfected with the following expression vectors: empty vector (Vector), PDK1 (WT and KD), or PDK1 siRNAs (#1 and #2, each 200 nm) in the presence of ASK1. The cells were then treated with or without 2 mm H₂O₂ for 30 min. ASK1 phosphorylation was determined by immunoblot analysis using anti-phospho-ASK1(Ser⁸³), anti-phospho-ASK1(Thr⁸⁴⁵), and anti-phospho-ASK1(Ser⁹⁶⁷) antibodies. D, parental HEK293 cells or HEK293 cells (PDK1 shRNA) stably expressing PDK1-specific shRNA were incubated for 30 min in the presence or absence of a specific Akt inhibitor VIII (20 μm). Cell lysates were examined for ASK1 phosphorylation (Ser⁸³, Thr⁸³⁸, and Ser⁹⁶⁷) by immunoblot analysis using the antibodies indicated. The knockdown level of endogenous PDK1 was determined by anti-PDK1 immunoblotting (fourth panel). The relative level of phosphorylation was quantitated by densitometric analyses, and the -fold increase relative to untreated control cells expressing an empty vector alone in the presence of ASK1 or parental cells was calculated. The circled letters P indicate phosphorylation. WB, Western blot; IP, immunoprecipitation.

FIGURE 5. — PDK1-mediated inhibition of ASK1 activity is dependent on the modulation of the association between ASK1 and its regulators, 14-3-3 and MKK3, and ASK1 phosphorylation. A and B, modulation of the endogenous association of ASK1 with 14-3-3 protein and MKK3. HEK293 cells were transfected with the indicated combinations of FLAG-14-3-3, FLAG-ASK1, HA-MKK3, HA-ASK1, Myc-tagged PDK1 (WT and KD), GST-PDK1, and PDK1-specific siRNA. Cell lysates were then subjected to immunoprecipitation with the antibodies indicated, and the resulting immunoprecipitates were analyzed by immunoblot analysis using the appropriate antibodies to determine the association of 14-3-3 protein (A) and MKK3 (B) with ASK1. The knockdown effect of endogenous PDK1 on the association between endogenous ASK1 and 14-3-3 protein or MKK3 was determined by immunoblot analysis with the antibodies indicated using HEK293 cells transiently transfected with PDK1-specific siRNA 1 (A and B, right panels). C, HEK293 cells were transiently transfected with the following expression vectors: empty vector (Vector), PDK1 (WT and KD), or PDK1 siRNAs (#1 and #2, each 200 nm) in the presence of ASK1. The cells were then treated with or without 2 mm H₂O₂ for 30 min. ASK1 phosphorylation was determined by immunoblot analysis using anti-phospho-ASK1(Ser⁸³), anti-phospho-ASK1(Thr⁸⁴⁵), and anti-phospho-ASK1(Ser⁹⁶⁷) antibodies. D, parental HEK293 cells or HEK293 cells (PDK1 shRNA) stably expressing PDK1-specific shRNA were incubated for 30 min in the presence or absence of a specific Akt inhibitor VIII (20 μm). Cell lysates were examined for ASK1 phosphorylation (Ser⁸³, Thr⁸³⁸, and Ser⁹⁶⁷) by immunoblot analysis using the antibodies indicated. The knockdown level of endogenous PDK1 was determined by anti-PDK1 immunoblotting (fourth panel). The relative level of phosphorylation was quantitated by densitometric analyses, and the -fold increase relative to untreated control cells expressing an empty vector alone in the presence of ASK1 or parental cells was calculated. The circled letters P indicate phosphorylation. WB, Western blot; IP, immunoprecipitation.