Protein a or g sepharose beads

Macrophages from WT, TLR2^−/− or TLR4^−/− mouse were incubated with Rv1016c (20 μg/ml) for 6 h and lysed with RIPA lysis buffer (Sangon, China). The lysates were pre-cleared by adding protein A or G sepharose beads (Santa Cruz, CA, USA) for 2 h. After centrifugation at 10,000 × g for 5 min at 4°C, the supernatant was incubated with Isotype IgG, anti-TLR2 or anti-His overnight at 4°C. After harvested and washed, the beads were boiled in 5x sample buffer for 5 min. The proteins were separated on 10% SDS-PAGE and probed with anti-TLR2 (BioLegend, CA, USA), or anti-His Abs (Santa Cruz, CA, USA) as indicated, followed by incubation with HRP-conjugated mouse anti-rat or rabbit anti-mouse IgG Abs. Immunoreactive bands were detected using an ECL reagent (Thermo Fisher Scientific, MA, USA) and visualized by exposure to x-ray film.

RAW 264.7 cells (2.0 × 10⁶/well) were treated with Rv2029c (10 μg/ml) for 8 h and lysed with RIPA buffer (Sangon Biotech, Shanghai, China). The resulting lysates were precleared by incubating with protein A or G sepharose beads (Santa Cruz Biotechnology, Dallas, TX, USA) for 2 h. After centrifugation at 10,000 × g for 5 min at 4°C, supernatants were incubated with isotype IgG, anti-TLR2, anti-TLR12, or anti-Rv2029c antibodies overnight at 4°C. The beads were then harvested, washed, and boiled in 5 × sample buffer for 5 min. Proteins were separated via 10% SDS-PAGE and transferred electrophoretically to PVDF membranes (Millipore, Billerica, MA, USA). After washing with TBST (Tris-buffered saline containing 0.5% Tween-20) buffer, membranes were probed with anti-TLR2, anti-TLR12 (BioLegend), or anti-His (Santa Cruz Biotechnology) antibodies, as indicated, followed by horseradish peroxidase (HRP)-conjugated mouse anti-rat or rabbit anti-mouse IgG. Immunoreactive bands were detected using an enhanced chemiluminescent (ECL) reagent (Thermo Fisher Scientific) and visualized by exposure to x-ray film.