Figure 1. Immunoblot of anti-pS83 ASK1 antibodies shows specificity for phosphorylated human ASK1. Anti-pS83 (aa 76-87) antibody, generated by immunization with phospho peptide coupled to KLH, was tested by immunoblot against lysates of Cos-7 cells after transient transfection, separately, with 1) vector only, 2) recombinant HA-ASK1, and 3) recombinant human HA-ASK1 where S83 was substituted with an alanine residue. Cells were lysed 24 h post-transfection in 200 microL of 1x SDS-sample buffer, heated at 96°C for 5, and vortexed for 30 sec. Samples (10 microL each) were separated on a 12% SDS-PAGE gel and transferred to PVDF (Millipore) followed by blocking for 45 using TTBS supplemented with 5% non-fat dry milk. All incubations were performed at room temperature. In panel a) all samples were incubated with 10 ug/mL mouse anti-HA antibody for 45. After 5X washes with TTBS, reaction with ALP rabbit anti-mouse IgG at 200 ng/mL proceeded for 45 following again by washing as before. The blot was developed using BCIP/NBT. This blot demonstrates both recombinant transfections were successfully over-expressed in the Cos-7 cells. In panel b) all samples were incubated with a 1:1,000 dilution of ASK1 antibody for 45. The antibody was pre-incubated with non-phospho peptide prior to membrane incubation. After 5X washes with TTBS, reaction with HRP goat anti-rabbit IgG at 10 ng/mL proceeded for 45 following again by washing as before. The membrane was processed as before. Lane 2 shows strong specific staining of ASK1. Lane 3, where S83 was replaced with alanine, shows greatly diminished staining. In panel c) all samples were incubated with a 1:1,000 dilution of ASK1 antibody as before except the antibody was preincubated with phospho peptide prior to membrane incubation. No staining is observed after phospho peptide blocking occurs.