Syk Phosphorylation

Syk phosphorylation assays were performed by immobilizing vascular cell adhesion molecule-1 (VCAM)-1 (3 ug/ml), CS-1-BSA (10 ug/ml), or specific monoclonal antibodies (anti-FcγRI mAb 276426, anti-α4β1 mAb 19H8, anti-β2 mAb 76C3, anti-αvβ3 mAb LM609 all at 5 ug/ml) overnight at 4°C in Tris-HCl pH 7.5, 150 mM NaCl, in 6-well plates (non-tissue culture treated, Becton Dickenson, cat# {"type":"entrez-nucleotide","attrs":{"text":"BD351146","term_id":"92252283","term_text":"BD351146"}}BD351146). Plates were then blocked with BSA (2% w/v) for 1 h at room temperature. In Figure 1C , GAM was immobilized in all wells (2 h at 37°C in 200 mM Na₂HCO₃ pH 9.2, blocked for 1 h in 2% BSA), followed by the addition of Poly-L-Lysine (PLL) or indicated monoclonal antibodies (5 ug/ml) in Tris-buffered saline (TBS) overnight at 4°C. After washing wells in serum-free RPMI, untreated or drug-loaded THP-1 cells (5 × 10⁶ cells per well) were plated in standard serum-free RPMI (1.5 ml total volume), allowed to adhere for 10 min at 37°C 5% CO_2, and then immediately lysed in RIPA buffer (1% Triton X-100, 50 mM Tris-HCl pH 7.2, 150 mM NaCl, 0.25% NaDOC, 0.05% SDS, 2 mM PMSF, 1 mM NaF, 1 mM Na₃VO₄, and protease inhibitor cocktail) (AEBSF, Aprotinin, E-64, Bestatin, Leupeptin and Pepstatin; VWR Cat#97063-010). Cells kept in suspension served as non-adherent controls and were lysed under the same conditions. For Syk immunoprecipitations, 5 × 10⁶ THP-1 cell equivalents were incubated overnight at 4°C with monoclonal antibody 4D10 at 4 ug/mg protein and were rotated. After overnight incubation, protein G-sepharose (20 ul packed beads washed in RIPA) (GE Healthcare cat# 17061801) was added to lysates, which were incubated for 2 h at 4°C, while rotating. Sepharose was washed 3× in RIPA by centrifugation, pelleted, and then resuspended in SDS-PAGE sample buffer (reducing). For whole cell lysates not subjected to immunoprecipitation, approximately 1 × 10⁶ cell equivalents were loaded for SDS-PAGE under reducing conditions (4–20%, Thermo Scientific, cat#25204). After electrophoresis, proteins were transferred to nitrocellulose for 1 h 100 V room temperature, blocked in 5% non-fat milk in TBS-T, and probed with the indicated primary antibody at 1:1,000 overnight at 4°C, followed by species-appropriate HRP secondary antibody (Southern Biotech) at 1:5,000 (30 min at room temperature), and detected using Pierce West Pico (Thermo Scientific cat# 34580). For blot stripping, nitrocellulose was subjected to 0.4 N NaOH for 20 min at room temperature, blocked as described above, and re-probed with the indicated antibodies (primary antibody at 1:1,000 for 1 h at room temperature, followed by HRP-conjugated secondary antibody at 1:5,000 for 30 min at room temperature).