Anti-ASK-1 phospho specific pS83 Antibody - Western Blot. Western blot of anti-pS83 ASK1 antibodies shows specificity for phosphorylated human ASK1. Anti-pS83 (aa 76-87) antibody was tested by western blot against Cos-7 cell lysates after transient transfection 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 ul of 1x SDS-sample buffer, heated at 96?C for 5, and vortexed for 30 sec. Samples (10 ul 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 anti-HA antibody. This blot demonstrates both recombinant transfections express rASK1. In panel b) all samples were incubated with anti-pS83 ASK1. 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 anti-pS83 ASK1 antibody as before except the antibody was pre-incubated with phospho peptide prior to membrane incubation. No staining is observed after phospho peptide blocking occurs.