Recombinant protein g sepharose 4b

RPMI-1640, Ammonium-Chloride-Potassium (ACK) lysing buffer, EDTA 0.5M, and HEPES 1M were purchased from Lonza (Walkersville, MD). MEM nonessential amino acids (NEAA), L-glutamine, Penicillin/streptomycin, Hanks’ Balanced Salt Solution with Ca²⁺ and Mg²⁺ (HBSS⁺) or HBSS without Ca²⁺ and Mg²⁺ (HBSS⁻), and Phosphate Buffered Saline with (PBS⁺) or without Ca²⁺ and Mg²⁺ (PBS⁻) were purchased from Corning Cellgro, Mediatech Inc. (Tewksbury, MA). Fetal bovine serum (FBS) was from Atlanta Biologicals (Oakwood, GA). Leukotriene B₄ (LTB₄) was from Cayman Chemical (Ann Arbor, MI). N-Formylmethionyl-leucyl-phenylalanine (fMLF), DNase I, Histopaques-1077 and −1119, 2,2'-azino-di-(3-ethyl)di-thiazoline-sulfonic-acid (ABTS), DuoLink^® In situ-Fluorescence kit, and proteinase inhibitor cocktail (cat#P8340) were from Millipore-Sigma (St. Louis, MO). Recombinant Protein G-Sepharose^® 4B, Hoechst 33342, CountBright counting beads, and Prolong antifade mounting agent were from Invitrogen (Carlsbad, CA). Recombinant murine E-selectin-Fc, VCAM-1-Fc and ICAM-1-Fc chimera molecules, and the polyclonal goat anti-mouse CD47 (cat#AF1866) and goat anti-human JAM-A (cat#AF1077) antibodies were from R&D Systems (Minneapolis, MN). Recombinant mouse TNF-α and IFN-γ were purchased from Peprotech (Rocky Hill, NJ). A complete list of antibodies used in this work is provided as supplemental material.

Cells for protein analysis were lysed in RIPA buffer (20 mM Tris-HCl, 37 mM NaCl₂, 2 mM EDTA, 1% Triton X-, 10% glycerol, 0.1% SDS, and 0.5% sodium deoxycholate) with phosphatase and protease inhibitors. For immunoprecipitations, cells were lysed in IP lysis buffer (100 mM NaCl, 25 mM Tris, 1% Triton X-, 10% glycerol, with phosphatase and protease inhibitors) and immunoprecipitated with 25 μL of 50% slurry of protein Recombinant protein G-Sepharose 4B (Thermo Fisher). Immunoprecipitates were washed three times with lysis buffer, once with high salt (500 mM NaCl), and once more with lysis buffer. Protein concentration in lysates was determined by using Protein Assay Kit (Bio-Rad). Cell extracts and immunoprecipitated proteins were denatured, subjected to SDS-PAGE, transferred to PVDF membranes (GE Healthcare). After blocking with 5% nonfat dry milk in Tris-buffered saline and 0.1% Tween (TBS-T), the membranes were incubated with the specific antibodies (as listed in key resources table) overnight at 4°C. After 2 h incubation with the appropriate horseradish peroxidase-conjugated antibodies, the immune complexes were detected by chemiluminescence (Thermo Fisher) or Near-infrared fluorescence (LI-COR).