Three sets of samples were prepared for each cell type to investigate the interaction with S. aureus. For the first set, THP-1 cells and HaCaT cells were infected with S. aureus in RPMI 1640 and DMEM media, respectively. The second set comprised THP-1 and HaCaT cells cultured alone in their respective growth media. The third set consisted of S. aureus cultured independently under identical conditions to those of the THP-1 and HaCaT cells (Fig. 1A). Following infection, cells were scraped off the plates into ice-cold lysis buffer (25 mM Tris HCl, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, and 5% glycerol) with protease and phosphatase inhibitors (Pierce Protease and Phosphatase Inhibitor Mini Tablets, EDTA-free, Thermo Fisher). The cell suspension was transferred into 2-mL screw-cap tubes containing 100 µL of 0.1-mm glass beads and lysed with bead-beating. The cell lysates were centrifuged for 20 min at 4°C and 14,000 rpm. The supernatant (total lysate) was transferred to a new tube. The lysis buffer was exchanged for the reaction buffer (4 M urea + lysis buffer) using the Zeba Spin Desalting Columns (Thermo Fisher). Following buffer exchange, the lysate protein concentration was measured using the Qubit protein assay kit, and the protein concentration was adjusted to 2 mg/mL using the reaction buffer. Each sample was mixed with 10 µL of 1 M MgCl2 and incubated for 1 min at room temperature. The ActivX desthiobiotin-ATP probe (Thermo Fisher) was reconstituted in ultrapure water to 20 µM, and 10 µL was added to each sample followed by incubation for 10 min at room temperature. Following labeling, 50 mL of 50% high-capacity streptavidin agarose resin slurry was added to each sample and incubated for 1 hour at room temperature with constant mixing on a rotator. The samples were centrifuged at 100 × g for 1 min to pellet the resin. The resins were washed two times with 500 mL of the reaction buffer, and the bound protein was eluted by adding 2× Laemmli reducing sample buffer and boiled for 5 min.
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