Journal: Molecular and Cellular Biology

Article Title: Neutrophils Stimulated with a Variety of Chemoattractants Exhibit Rapid Activation of p21-Activated Kinases (Paks): Separate Signals Are Required for Activation and Inactivation of Paks

doi: 10.1128/mcb.18.12.7130

Figure Lengend Snippet: FIG. 1. Effects of various agonists on the activation of the 63- and 69-kDa Paks in neutrophils. Autoradiographs demonstrate the ability of 1.0 mM fMLP (A), 1.0 mM PAF (B), 20 nM LTB4 (C), 12.5 nM IL-8 (D), 65 nM RANTES (E) and 100 mg of sulfatide per ml (F) to trigger activation of the 63- and 69-kDa Paks in neutrophils. Paks were monitored by the ability to undergo renaturation and catalyze the phosphorylation of the p47-phox peptide fixed in a gel. Cells were treated with solvent/vehicle for 15 s (i.e., unstimulated cells) (lane a), agonist for 15 s (lane b), agonist for 30 s (lane c), agonist for 1.0 min (lane d), agonist for 3.0 min (lane e), and solvent/vehicle for 3.0 min (lane f). Positions of the 63- and 69-kDa Paks are designed by arrows and arrowheads, respectively.

Article Snippet: Recombinant human interleukin-8 (IL-8) and recombinant human RANTES (acronym for “regulated upon activation, normal T-cell expressed and presumably secreted”) were purchased from R&D Systems Inc., Minneapolis, Minn. An affinity-purified rabbit polyclonal antibody (Ab) raised against a peptide corresponding to amino acid residues 525 to 544 of rat Pak1 [a Pak(C-19) Ab] along with Abs to Pak2 [g Pak(V-19) Ab] and Pak3 [b Pak(L-18) Ab] were purchased from Santa Cruz Biotechnology, Inc., Santa Cruz, Calif. Affinity-purified rabbit polyclonal Abs that recognize the active (doubly phosphorylated) forms of JNK and p38-MAPK were obtained from Promega Corporation, Madison, Wis.

Techniques: Activation Assay, Phospho-proteomics, Solvent

Journal: Molecular and Cellular Biology

Article Title: Neutrophils Stimulated with a Variety of Chemoattractants Exhibit Rapid Activation of p21-Activated Kinases (Paks): Separate Signals Are Required for Activation and Inactivation of Paks

doi: 10.1128/mcb.18.12.7130

Figure Lengend Snippet: FIG. 2. Activation of the 63- and 69-kDa Paks in neutrophils treated with different concentrations of agonists. Neutrophils were stimulated with different amounts of the indicated agonists for 15 s, and the 63- and 69-kDa Paks were assayed as described in Materials and Methods. In lanes a and b of all panels, cells were also treated for 15 s with 0.25% (vol/vol) Me2SO (i.e., unstimulated cells) and 1.0 mM fMLP for comparative purposes, respectively. (A) The con- centrations of PAF in lane a and in lanes c through h were 0.0 mM (0.25% [vol/vol] Me2SO), 5.0 mM, 1.0 mM, 0.10 mM, 50 nM, 10 nM, and 1.0 nM, respectively. (B) The concentrations of LTB4 in lanes c through i were 0.0 mM (0.25% [vol/vol] ethanol), 0.50 mM, 0.10 mM, 20 nM, 5.0 nM, 1.0 nM, and 0.10 nM. (C) The concentrations of IL-8 in lanes c through i were 0.0 mM (0.001% [wt/vol] BSA in PBS), 0.25 mM, 0.125 mM, 12.5 nM, 6.3 nM, 1.3 nM, and 0.13 nM. (D) The concentrations of RANTES in lanes c through i were 0.0 mM (0.001% [wt/vol] BSA in PBS), 0.13 mM, 65 nM, 13 nM, 6.5 nM, 1.3 nM, and 0.65 nM. (E) The concentrations of sulfatide in lanes c through i were 0.0 mM (PBS), 400 mg/ml, 200 mg/ml, 100 mg/ml, 50 mg/ml, 10 mg/ml, and 1.0 mg/ml. Positions of the 63- and 69-kDa Paks are designated by arrows and arrowheads, respectively.

Article Snippet: Recombinant human interleukin-8 (IL-8) and recombinant human RANTES (acronym for “regulated upon activation, normal T-cell expressed and presumably secreted”) were purchased from R&D Systems Inc., Minneapolis, Minn. An affinity-purified rabbit polyclonal antibody (Ab) raised against a peptide corresponding to amino acid residues 525 to 544 of rat Pak1 [a Pak(C-19) Ab] along with Abs to Pak2 [g Pak(V-19) Ab] and Pak3 [b Pak(L-18) Ab] were purchased from Santa Cruz Biotechnology, Inc., Santa Cruz, Calif. Affinity-purified rabbit polyclonal Abs that recognize the active (doubly phosphorylated) forms of JNK and p38-MAPK were obtained from Promega Corporation, Madison, Wis.

Techniques: Activation Assay

Journal: Molecular and Cellular Biology

Article Title: Neutrophils Stimulated with a Variety of Chemoattractants Exhibit Rapid Activation of p21-Activated Kinases (Paks): Separate Signals Are Required for Activation and Inactivation of Paks

doi: 10.1128/mcb.18.12.7130

Figure Lengend Snippet: FIG. 11. Effects of a variety of agonists on the activation of JNK and p38- MAPK in neutrophils. Activation of JNK and p38-MAPK was monitored in neutrophils by Western blotting with antibodies that recognized only the acti- vated (doubly phosphorylated) forms of these kinases. Stimulation of the cells and Western blotting were performed as described in Materials and Methods. Membranes were first blotted with an antibody to activated JNK (arrows) and then reblotted with an antibody to activated p38-MAPK (arrowheads). Neutro- phils were treated for 3 min at 37°C with 0.25% (vol/vol) Me2SO (unstimulated cells) (lane a), 1.0 mM fMLP (lane b), 1.0 mM PAF (lane c), 25 nM C5a (lane d), 20 nM LTB4 (lane e), 65 nM RANTES (lane f), 100 mg of sulfatide per ml (lane g), and 12.5 nM IL-8 (lane h). The broken arrow shows the position of an unknown protein which also reacted with the antibody to activated p38-MAPK.

Article Snippet: Recombinant human interleukin-8 (IL-8) and recombinant human RANTES (acronym for “regulated upon activation, normal T-cell expressed and presumably secreted”) were purchased from R&D Systems Inc., Minneapolis, Minn. An affinity-purified rabbit polyclonal antibody (Ab) raised against a peptide corresponding to amino acid residues 525 to 544 of rat Pak1 [a Pak(C-19) Ab] along with Abs to Pak2 [g Pak(V-19) Ab] and Pak3 [b Pak(L-18) Ab] were purchased from Santa Cruz Biotechnology, Inc., Santa Cruz, Calif. Affinity-purified rabbit polyclonal Abs that recognize the active (doubly phosphorylated) forms of JNK and p38-MAPK were obtained from Promega Corporation, Madison, Wis.

Techniques: Activation Assay, Western Blot

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Cutting edge: T cells trigger CD40-dependent platelet activation and granular RANTES release: a novel pathway for immune response amplification.

doi: 10.4049/jimmunol.172.4.2011

Figure Lengend Snippet: FIGURE 3. CD40-dependent RANTES release by platelets. RANTES re- lease by soluble and membrane-bound CD40-stimulated platelets. Platelets were stimulated either with sCD40L or paraformaldehyde-fixed D1.1 cells (1: 10), and RANTES content in the supernatants was measured by ELISA. Data are mean SEM of three separate experiments. , p 0.01 for sCD40L- and D1.1 cell-stimulated platelets vs blocking Ab treatment.

Article Snippet: RANTES ELISA kits and human recombinant RANTES were purchased from R&D Systems (Minneapolis, MN) and phospho-p38 and phospho-extracellular signalregulated kinase (ERK) Abs from Cell Signaling (Beverly, MA).

Techniques: Membrane, Enzyme-linked Immunosorbent Assay, Blocking Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Cutting edge: T cells trigger CD40-dependent platelet activation and granular RANTES release: a novel pathway for immune response amplification.

doi: 10.4049/jimmunol.172.4.2011

Figure Lengend Snippet: FIGURE 4. Involvement of p38 MAP kinase in platelet CD40 signaling. A, Immunoblotting of CD40-induced p38 phosphorylation in sCD40L-stimulated platelets. Platelets were stimulated with sCD40L and then lysed at the indicated time points. Immunoblotting was performed with Abs specific for phosphorylated p38 and ERK. This figure is representative of three separate experiments. B, Inhi- bition of CD40-dependent platelet activation by SB203580. Platelets were acti- vated with sCD40L with or without pretreatment with the p38 MAP kinase inhib- itor SB203580 and then submitted to flow cytometric analysis for P-selectin expression. The black curve represents the background signal from the isotype con- trol. The presence of DMSO, used for SB203580 dilution, had no effect on P- selectin expression. Data represent the mean SEM of three separate experiments. , p 0.01 for inhibitor-treated compared with sCD40L-stimulated platelets. C, Inhibition of CD40-dependent RANTES release by SB203580. Platelets were stimulated with sCD40L in the presence and absence of SB203580, and RANTES content in the supernatants was measured by ELISA. Data are mean SEM of three separate experiments. , p 0.01 for inhibitor-treated compared with sCD40L-stimulated platelets.

Article Snippet: RANTES ELISA kits and human recombinant RANTES were purchased from R&D Systems (Minneapolis, MN) and phospho-p38 and phospho-extracellular signalregulated kinase (ERK) Abs from Cell Signaling (Beverly, MA).

Techniques: Western Blot, Phospho-proteomics, Activation Assay, Inhibition, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Cutting edge: T cells trigger CD40-dependent platelet activation and granular RANTES release: a novel pathway for immune response amplification.

doi: 10.4049/jimmunol.172.4.2011

Figure Lengend Snippet: FIGURE 5. Platelet-derived RANTES mediates increased T cell recruitment. A, Detection of platelet-derived and recombinant RANTES on the surface of HIMEC by confocal microscopy. Confluent HIMEC monolayers were exposed to degranulated platelet-derived or recombinant (r) RANTES and fluorescently labeled for detection of RANTES (Alexa 488 secondary Ab, green) and nuclei (DAPI, blue). This figure is representative of four separate experiments. B, Platelet-derived RANTES-mediated T cell adhesion to HIMEC and inhibition by met-RANTES. HIMEC monolayers were left untreated (baseline) or exposed to degranulated platelet-derived or recombinant RANTES. MOLT4 cells, preincubated or not with met-RANTES, were added to the HIMEC monolayers. The number of adherent cells in each experimental condition was expressed as mean SEM of four separate experiments. , p 0.05 for met-RANTES-pretreated compared with untreated MOLT4 cells.

Article Snippet: RANTES ELISA kits and human recombinant RANTES were purchased from R&D Systems (Minneapolis, MN) and phospho-p38 and phospho-extracellular signalregulated kinase (ERK) Abs from Cell Signaling (Beverly, MA).

Techniques: Derivative Assay, Recombinant, Confocal Microscopy, Labeling, Inhibition