Nuclear extracts were prepared from monocytes as previously described (57). Briefly, harvested and pelleted monocytes were incubated in 400 μl of cold buffer A [10 mM Hepes (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 10 mM EGTA, 1 mM dithiothreitol (DTT), and protease inhibitors (PI)] on ice for 15 min followed by addition of 25 μl of 10% NP-40 followed by vortexing and centrifugation at 15,000 rpm for 1 min. The supernatant was removed, and the nuclear pellet was resuspended in ice-cold buffer C [20 mM Hepes (pH 7.9), 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, and PI], incubated on ice with vigorous shaking for 15 min, and centrifuged at 15,000 rpm for 1 min. The supernatant was harvested, and proteins were resolved using 7.5% TGX precast gel (Bio-Rad).

THP-1 cells were lysed in 1% NP-40 lysis buffer on ice for 30 min. Total cell lysates were resolved using 6% TGX Novex gel (Invitrogen).

Tc CM, M8I-treated Tc CM, or NSTc CM were concentrated 3- to 18-fold using a 50-kDa MWCO Centrifugal Filter Unit, mixed 1:1 with 2× sample buffer (Bio-Rad, catalog no. 161-0737), and boiled for 5 min. Human platelets were lysed in 1% NP-40 lysis buffer (Invitrogen) on ice for 30 min. Concentrated Tc CM, NSTc CM, M8I-treated Tc CM, platelet extracts, and mouse NS0 myeloma cell line–derived human rGPIbα protein (120 kDa; R&D systems, catalog no. 4067-GP-050) were resolved using 7.5% TGX precast gel (Bio-Rad).

After SDS-PAGE, the proteins were transferred to polyvinylidene difluoride membranes and probed with the following antibodies: rabbit anti–NF-κB p65 (1:1000; Cell Signaling Technology, catalog no. 8242 or 4764); mouse anti-αM integrin (CD11b/CD42b) (1:1000; Santa Cruz Biotechnology, catalog no. sc-515923); mouse anti–TATA-binding protein (1:2000; catalog no. ab818) and mouse HRP–anti-GAPDH (1:30,000; catalog no. ab9482) (all from Abcam); and donkey HRP–anti-rabbit and sheep HRP–anti-mouse (1:10,000; both from GE Healthcare). The GPIbα protein was detected using mouse anti-CD42b Ab clone SZ2 that recognizes the sulfated tyrosine sequence (epitope 269-282) in GPIbα (Santa Cruz Biotechnology, catalog no. sc-59052) followed by biotin anti-mouse IgG1 rabbit mAb clone RM106 (1:5000; RevMAb Biosciences, catalog no. 31-1002-02) and streptavidin-HRP (1:10,000; Abcam, catalog no. ab7403). Densitometry was performed using Gel Doc XR+ Gel Documentation System (Bio-Rad).

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