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. 2003 Jun 15;111(12):1933–1943. doi: 10.1172/JCI17790

Figure 6.

Figure 6

AIP1 binds preferentially to dephosphorylated ASK1 at Ser-967. (a) The 14-3-3 binding site (pSer-967) in ASK1 is not critical for AIP1 association. AIP1 was cotransfected with vector control, ASK1, or ASK1-S967A, a mutant defective in 14-3-3 binding. Interaction of AIP1 and ASK1 was examined by immunoprecipitation with anti-ASK1 followed by Western blot with anti-FLAG. S/A, ASK1-S967A. (b) Phosphatase treatment increases AIP1-ASK1 complex. Cell lysates containing ASK1 and AIP1-N were incubated with alkaline phosphatase (10 U PPase/40 μg cell lysate) at room temperature for 1 hour. Treated lysates were used IP by anti-ASK1, followed by Western blot with anti-FLAG. A GST–14-3-3 pull-down assay was used as a control for phosphatase treatment. Bound ASK1 was detected by Western blot with anti-FLAG. (c) Peptide specificity. ASK1 was used in a GST–14-3-3 pull-down assay in the presence of peptide ASK1-S or ASK1-pS (0.3 mM). Bound ASK1 was detected by Western blot with anti-FLAG. (d) Peptide competition assay for AIP1-ASK1 interaction. Cell lysates containing ASK1 and AIP1-N were used for immunoprecipitation assay as described in a in the presence of peptide ASK1-S or ASK1-pS (0.3 mM). Interaction of AIP1 and ASK1 was examined by immunoprecipitation with anti-ASK1, followed by Western blot with anti-FLAG.