TSP-4 variants activate different protein tyrosine phosphorylation pathways in adherent PMNs. (A) PMNs were stimulated with PMA (1 nM) and seeded onto plates coated with TSP-4 variants (80 nM) and control PVP and incubated for up to 2 hours at 37°C. After washing, adherent cells were lysed. Equal amounts of total protein in cell lysates were analyzed by Western blot with antiphosphotyrosine mAb. (B) PMNs were treated as described in panel A. Cell lysates were analyzed by Western blot with Abs to phospho-p38 MAPK and total p38 MAPK. (C) Adhesion assay was performed as described in Figure 3A with one exception: cells were incubated for 30 minutes at 37°C. Adherent cells were stained with Ab to phosphorylated p38 MAPK, followed by incubation with FITC-conjugated goat anti–rabbit IgG Ab. Cells were analyzed as described in “Materials and methods.” Bar size, 10 μm. Images captured using a 40 ×/0.7 NA objective lens. (D) The cells were treated as described in panel A, and PMN lysates were analyzed by Western blot using PathScan Multiplex Western cocktail III containing Abs to phosphorylated forms of signal transducer and activator of transcription-1 (STAT1), c-Jun NH2-terminal kinase (JNK), S6 ribosomal protein, and heat shock protein 27 (HSP27).