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Blood, Vol. 109, Issue 1, 176-184, January 1, 2007
Phospholipase C, calcium, and calmodulin are critical for  4ß1 integrin affinity up-regulation and monocyte arrest triggered by chemoattractants
Blood Hyduk et al.
109: 176
Supplemental materials for: Hyduk et al, Vol 109, Issue 1, 176-184
SDS-PAGE and Western blotting Cells were resuspended at 20 × 106/mL in assay buffer in the presence or absence of appropriate pharmacologic inhibitors as indicated in “Materials and methods.” Cells were stimulated with 12.5 nM SDF-1 or 100 nM fMLP prior to the addition of an equal volume of ice-cold lysis buffer. Cell lysis buffer (2×) contained 40 mM Tris, 300 mM NaCl, 20 mM EDTA, 2 mM sodium orthovanadate, 2 mM NaF, 2% Triton X-100, 0.1% Tween 20, 1 mM PMSF, and other protease inhibitors (complete mini protease inhibitor cocktail; Roche, Laval, QC, Canada). Lysates were centrifuged at 12 000g for 15 minutes at 4°C to remove nuclei and insoluble cytoskeleton. Protein concentration in lysate supernatants was determined using a DC-protein assay kit (Bio-Rad, Mississauga, ON, Canada). Lysates from unstimulated Jurkat cells were used as positive controls where indicated. Lysates were separated on 8% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gels (100 V, 22°C) and electrophoretically transferred to polyvinylidene difluoride membrane (22 V, 4°C, 18 hours). Membranes were probed with primary Abs and developed using HRP-conjugated secondary Abs and the enhanced chemiluminescence (ECL) system (Amersham Pharmacia Biotech, Baie d’Urfé, QC, Canada). Antibodies included anti–phospho-Thr202/Tyr204 p44/42 MAPK (E10), rabbit polyclonal anti-p44/42 MAPK, rabbit polyclonal anti–phospho-Ser473 AKT, mouse monoclonal anti-AKT, anti–phospho-MAPKAP2, and rabbit polyclonal anti-MAPKAP2 from Cell Signaling Technologies. Individual blots were stripped in 100 mM 2-ME, 2% SDS, and 62.5 mM Tris-HCl (pH 6.7) prior to reprobing with a second antibody.
Files in this Data Supplement:
- Table S1. α4 Integrin affinity on monocytes is rapidly and transiently up-regulated by a broad range of stimuli (PDF, 17.9 KB) -
LDV-binding to human peripheral blood monocytes was measured by flow cytometry following stimulation by a range of agonists. All chemokines were used at 12.5 nM. These stimuli, which trigger a G proteinencoupled Ca2+i flux in monocytes, induced a rapid and transient increase in LDV-FITC binding.
- Figure S1. p44/42 and p38 MAPK and PI3K are not required for fMLP- and SDF-1–induced LDV-FITC binding to U937-FPR cells (PDF, 2.77 MB) -
(A) Western blots of U937-FPR cells pretreated with DMSO, U0126, or SB203580 and stimulated for 1 minute with buffer (control), SDF-1, or fMLP. Blots were first probed for phospho-Thr202/Tyr204 p44/42 MAPK, then reprobed for total p44/42 MAPK, or phospho-MAPKAP2 followed by total MAPKAP2. One of 2 independent experiments is shown. (B) Binding of LDV-FITC to U937-FPR cells pretreated with DMSO (0.1%, vehicle control), U0126, or SB203580 was determined prior to and following the stimulation (at time 0) with fMLP (100 nM), SDF-1 (12.5 nM), Mn2+ (0.5 mM), or buffer. One of at least 4 independent experiments is shown. (C) Western blots of U937-FPR cells pretreated with DMSO, wortmannin (100 nM), or LY294002 (20 or 50 µM) and stimulated for 1 minute with buffer (control), SDF-1 (12.5 nM), or fMLP (100 nM). Blots were first probed for phospho-Ser473 AKT, then stripped and reprobed for total AKT. One of 2 independent experiments is shown. (D) Flow cytometry was used to determine LDV-FITC binding in real time to U937-FPR cells pretreated with DMSO, wortmannin, or LY294002 prior to addition of fMLP, SDF-1, Mn2+, or buffer at time 0. In the lower panel, Mn2+ was added 20 minutes prior to analysis. One of 3 independent experiments is shown.
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