Anti tlr2

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.

Cells were washed twice with PBS. Membrane and cytosol proteins were extracted according to the manufacturer’s instructions using the Qproteome Cell Compartment Kit (Qiagen, Hilden, Germany, Cat. No. 37502). Protein concentration was measured using the Bradford method. Extracted proteins were resolved in sodium dodecyl sulphate–polyacrylamide (SDS) gel electrophoresis under reducing conditions and then transferred to nitrocellulose. The blocking was done using TBST buffer (25 mm Tris-HCl, pH8·0, 125 mm NaCl, 0·1% Tween 20) containing 5% fat-free milk. Blots were incubated with anti-Actin, anti-TLR2, anti-TLR4, anti-SOCS1, anti-SHIP1 antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and horseradish peroxidase (HRP)-conjugated IgG. The membranes were visualized using SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, San Jose, CA, USA, Cat. No. 34080).

Recombinant M-CSF was purchased from Peprotech (Rocky Hill, NJ, USA). Fluorescein isothiocyanate (FITC)-annexin V/PI kits were purchased from BD Biosciences (BD Pharmingen, San Jose, CA). LPS from E. coli O111:B4 was purchased from InvivoGen (San Diego, CA, USA). Endotoxin filter (END-X) and endotoxin removal resin (END-X B15) were acquired from the Associates of Cape Cod (East Falmouth, MA, USA). Anti-phosphorylated ERK1/2 monoclonal Ab, anti-ERK1/2 monoclonal Ab, anti-phosphorylated p38 monoclonal Ab, anti-p38 monoclonal Ab, anti-phosphorylated JNK monoclonal Ab, anti-JNK monoclonal Ab, anti-phosphorylated IκB-α monoclonal Ab, anti-IκB-α monoclonal Ab, and anti-tubulin polyclonal Ab were obtained from Cell Signaling Technology (Danvers, MA, USA). Anti-TLR2, anti-TLR4, and anti-histidine (His) antibodies (Abs) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). HRP-conjugated anti-mouse IgG Ab and HRP-conjugated anti-rabbit Ab were obtained from Calbiochem (San Diego, CA, USA). Phycoerythrin (PE)-conjugated mAbs directed against IL-10, IL-12p70, CD80, CD86, and MHC class II, and allophycocyanin-conjugated mAb directed against F4/80 were purchased from eBioscience (San Diego, CA, USA). Mouse TNF-α, MCP-1, IL-6, IL-10, IL-12p70, IFN-γ, and IL-2 ELISA kits were obtained from eBioscience.