Biochemical analysis of cell signaling in response to surface-bound ICAM-1

Dishes (60 mm; 430166, Corning) were coated with ICAM-1 (2 μg/ml) overnight at 4°C. Activated CD4+ T cells were serum starved for 3 hours in DMEM, washed, and resuspended in CMF-HBS, followed by a wash in CMF-HBS containing 10 mM EDTA. T cells were then washed and resuspended in CMF-HBS and incubated for 20 min at 37°C. T cells were then treated with Ca2+ and Mg2+ (unstimulated control), treated with Mn2+ alone or together with soluble ICAM-1 (1 μg/ml), or treated with Mn2+ and allowed to interact with surface-bound ICAM-1. After 20 min at 37°C, cells stimulated in solution were lysed by adding 2× ice-cold lysis buffer (final lysis buffer composition: 1% Triton X-100, 150 mM NaCl, 50 mM Hepes, 10 mM MgCl2, 5 mM NaF, 1 mM sodium orthovanadate, and Roche EDTA-free protease inhibitor cocktail). Cells stimulated on surface-bound ICAM-1 were lysed by aspirating the medium and adding 1× ice-cold lysis buffer. Lysates were incubated on ice with periodic vortexing for 20 min, followed by centrifugation for 10 min at 16,000g at 4°C. Lysates were used for immunoprecipitations and GST pulldowns (see below). A small aliquot of each whole-cell lysate was retained, mixed with 4× sample buffer containing dithiothreitol (50 mM final), and heated to 95°C for 10 min before separation by SDS–polyacrylamide gel electrophoresis (SDS-PAGE).

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