Fig 1: Cath-Ka directly binds to both TLR2 and TLR4. (A) Flow cytometry measurement of the binding reaction of FITC-labeled Cath-Ka (1.25–20 µM) with RAW264.7 cells. (B) The effects of TLR inhibitors on Cath-Ka-induced TNF-α production. RAW264.7 cells were pre-treated with the indicated inhibitors for 30 min and then co-cultured with Cath-Ka (10 µM) for 24 h before TNF-α production was measured by ELISA. (C) The effects of Cath-Ka (10 µM) on LTA (10 µg/mL) - or LPS (100 ng/mL)- induced TNF-α production in RAW264.7 cells. (D) Representative SPRi sensograms of Cath-Ka binding to TLR2 or TLR4 immobilized on a gold chip. Recombinant human TLR2 and TLR4 (1 mg/mL) were immobilized on an activated bare gold SPRi chip before Cath-Ka (0.25–4 µM) was used as the mobile phase. (E) Representative WB images of TLR2 protein pulled down by Cath-Ka. TLR2 protein of RAW264.7 cell lysates was captured with biotin-tagged Cath-Ka-bound Streptavidin resin before it was eluted and used for WB analyses. Cath-Ka-1 was used as the negative control. (F) Representative WB images of CETSA. RAW264.7 cells were stimulated with Cath-Ka (10 µM) or PBS for 1 h under the indicated temperatures before TLR2 (a) and TLR4 (b) protein expressions were detected by WB. Data are expressed as mean ± SD (n = 3). ***P < 0.001 is thought statistically significant to the control group, while #P < 0.05, ##P < 0.01, and ###P < 0.001 are deemed to have significant differences to the corresponding Cath-Ka/LTA/LPS treatment groups; ns means not significant
Fig 2: Cath-Ka activates TLR2/4-MyD88-MAPKs signaling pathways in vitro and vivo. (A, B) Representative WB images (left) and quantitative analysis (right) of the expression of TLR2/4-MyD88-MAPKs signaling proteins activated by Cath-Ka. (A) RAW264.7 cells were stimulated with Cath-Ka (10 µM) for the indicated time (0.5, 1, 2, 4, and 6 h); (B) RAW264.7 cells were stimulated with the indicated concentrations of Cath-Ka (2.5, 5, 10, and 20 µM) for 4 h (for the detection of TLR2, TLR4, and MyD88) or 0.5 h (for the detection of MAPKs) before the proteins related to TLR2/4-MyD88-MAPKs signaling pathways were measured by WB. (C, D) Representative WB images (left) and quantitative analysis (right) of the effects of C29 and TAK-242 on the expression of proteins related to TLR2/4-MyD88-MAPKs signaling pathways in the presence of Cath-Ka. RAW264.7 cells were pre-treated with 10 µM C29 (C) or 10 µM TAK-242 (D) for 30 min and then were cultured with medium containing Cath-Ka (10 µM) for 4 h (for the detection of TLR2, TLR4, and MyD88) or for 0.5 h (for the detection of MAPKs) before the proteins of TLR2/4-MyD88-MAPKs signaling pathways were measured by WB. (E) Representative WB images (left) and quantitative analysis (right) of TLR2/4-MyD88-MAPKs signaling protein expression in peritoneal cells activated by Cath-Ka. Data are expressed as mean ± SD (n = 3–4). *P < 0.05, **P < 0.01, and ***P < 0.001 are thought statistically significant to the control group, while #P < 0.05, ##P < 0.01, and ###P < 0.001 are deemed to have significant differences between the corresponding Cath-Ka treatment groups; ns means not significant
Supplier Page from Abcam for Recombinant Human TLR2 protein (His tag C-Terminus)