Anti tlr2

C57BL/6 mouse macrophages were treated with PPE26 (10μg/ml) for 6 h and lysed with RIPA lysis buffer (Sangon, China). After pre-clearing with protein A/G sepharose beads (Santa Cruz, CA, USA) for 2 h, the mixture of cell lysates and bead was centrifuged at 10,000 x g for 5 min at 4°C. The supernatant was incubated with anti-TLR2, anti-TLR4 or anti-PPE26 overnight at 4°C, then the Ab-bound proteins were pull down using protein A /G beads for 6 h at 4°C. The beads were harvested, washed, and boiled in 5x sample buffer for 5 min. The proteins were separated on 10% SDS-PAGE and probed with anti-TLR2, anti-TLR4 (BioLegend, CA, USA), and anti-His Abs (Santa Cruz, CA, USA) as indicated, followed by incubation with HRP-conjugated mouse anti-rat or rabbit anti-mouse secondary IgG secondary Abs. Target bands were visualized using the ECL reagent (Thermo Fisher Scientific, MA, USA).

Dermal papilla cells (hDPCs) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; Hyclone) and antibiotic/antimycotic solution (Gibco BRL, Gaithersburg, MD, USA) containing penicillin and streptomycin. Cells were incubated at 37 °C in a 5% CO₂ incubator. All cultures used for experiments were in the third or fourth passage. Depending on the experiment, the media were supplemented with different concentrations of recombinant human HMGB1 (HMGB1; R&D systems, Minneapolis, MN, USA) or HMGB1 with antibodies blocking receptor for advanced glycation end-products (RAGE-FC, R&D systems), anti-TLR2 (BioLegend, San Diego, CA, USA), or anti-TLR4 (eBiosicence, San Diago, CA, USA) neutralizing antibodies. For the analysis on the effects of different forms of HMGB1, 200 ng/ml of A-box and 200 ng/ml of B-box (HMGBiotech, Milano, Italy), and 5 mM of dithiothreitol (DTT; Sigma–Aldrich, St. Louis, MO, USA) were used.

It is reported that “TLR2 is the major immune receptor involved in S. suis recognition” (39 (link), 40 (link)). To investigate which receptor was specifically responsible for HP1330-mediated cytokine upregulation, we first detected TLR2 changes after HP1330 stimulation by qRT-PCR, with TLR4 as a control. Second, antibody blocking assays were performed as previously described (41 (link)). After pretreatment with 8 µg of an anti-TLR2 (BioLegend) or anti-TLR4 (BioLegend) antibody for 30 min, RAW264.7 cells were incubated with 10 µg⋅ml⁻¹ HP1330 for 6 h. The concentrations of TNF-α, MCP-1, and IL-1β in the culture supernatants were determined by ELISA. On the basis of these experiments, we identified the recognition receptor of HP1330. Finally, TLR2−/− and TLR4−/− macrophages were isolated from TLR2−/− and TLR4−/− mice to verify the above results.

To investigate which receptor was specifically responsible for HP1717-mediated cytokine upregulation, we first detected the mRNA levels of TLR2 and TLR4 after HP1717 stimulation by qRT-PCR. Next, antibody blocking assays were performed to verify the results of qRT-PCR. Briefly, after pretreatment with 8 μg of an anti-TLR2 (BioLegend, San Diego, CA, USA) or anti-TLR4 (BioLegend) antibody for 30 min, RAW264.7 cells were incubated with 1 μg⋅mL⁻¹ HP1717 for 6 h. The concentrations of IL-1β, MCP-1, and TNF-α in the culture supernatants were determined by ELISA. TLR2−/− and TLR4−/− macrophages isolated from TLR2−/− and TLR4−/− mice were also used to prove this result.

C57BL6 mice were purchased from National Institute of Nutrition and maintained in a pathogen-free condition at NIAB animal facility. The animal care and all the experimental procedures were performed in accordance with the guidelines approved by Institutional Animal Ethics Committee (IAEC) of National Institute of Animal Biotechnology (NIAB). A mouse macrophage cell line (RAW264.7) human embryonic kidney cell line (HEK293) were originally purchased from the American Type Culture Collection (Manassas, VA). WT (NR- 9456), TLR2^−/−(NR-9457), TLR4^−/−(NR-9458), TLR2^−/−/4^−/− DKO (NR-19975) cell lines were obtained from BEI Resources, USA. Cells were cultured in DMEM (Sigma, USA) supplemented with 10% FBS (Invitrogen, Carlsbad, CA, USA), penicillin (100 U/ml), and streptomycin (100 mg/ml) and maintained at 37 °C in a humidified incubator (5% CO₂). Anti-TLR2 and anti-TLR4 were purchased from Biolegend, USA and the inhibitors NF-kB (SN50), p38 (SB203580) and JNK (SP600125) were from Invivogen and Sigma-Aldrich (St. Louis, MO). Human and mouse IL-8, IL-6 and TNF-α sandwich ELISA kits were from R&D biosystems. PerCP-CY5.5–conjugated anti MHC-II, PE-conjugated CD80, APC conjugated CD86 and BV421- CD40 for flow cytometry experiments were procured from BD biosciences, US. All reagents were purchased from Sigma unless otherwise specified.

Middlebrook 7H9, 7H10, and OADC were obtained from Difco (Detroit, MI). Annexin V-PE was from BD PharMingen (Mississauga, Ontario, Canada). OVA:I-Ab complexes were detected with DOBW T hybridoma cells (Pai et al., 2003 (link)). Anti-His was obtained from antibodies (Santa Cruz Biotechnology, SantaCruz, USA). Anti-Rv1016c, Anti-Rv2145c and Anti-Rv3425 mouse polyclonal antibody were from C57BL/6 mouse immunized by synthesized respective peptides in our lab. The primary Abs used included rabbit antiERK2, rabbit anti-p38, rabbit anti-JNK, rabbit anti-phosphoERK1/2, rabbit anti-phospho-p38, rabbit anti-phospho-JNK, rabbit anti-phospho-IκBα, and peroxidase (HRP)-conjugated secondary Abs (Cell Signaling Technology). Anti-TLR2 and anti-TLR4 were from BioLegend. Alexa488-conjugated anti- His Abs, Alexa488-conjugated or Alexa555-conjugated secondary antibodies were obtained from (Cell Signaling Technology).