Journal: Molecular Pain

Article Title: TLR3 mediates central sensitization in a chronic migraine model induced by repeated nitroglycerin through the ERK signaling pathway

doi: 10.1177/17448069251346373

Figure Lengend Snippet: TLR3 regulates the expression of c-Fos and CGRP by activating the ERK signaling pathway. (a–d) Immunoblot analysis was performed in the VEH, NTG, NTG+DMSO, and NTG+CU CPT4a groups for p-MEK1/2 to MEK1/2 ratio, p-ERK1/2 to ERK1/2 ratio, p-TAK1 to TAK1 ratio, and TRAF6 ( n = 5). (e–f) Immunoblot analysis was performed in the VEH, NTG, NTG+DMSO, and NTG+PD98059 groups for p-ERK1/2 to ERK1/2 ratio, CGRP and c-Fos ( n = 5). Data are the mean ± SEM; one-way ANOVA and Tukey’s post hoc tests. * p < 0.05, ** p < 0.01, *** p < 0.001 versus the VEH group, and # p < 0.05, ## p < 0.01, ### p < 0.001 versus the NTG group.

Article Snippet: Then, we placed the NC membrane in a TBST solution containing 5% skim milk powder and sealed it at room temperature for 1 h. Next step, we incubated it overnight with the following antibodies at 4°C: mouse anti-TLR3 (1:500, ab13915, Abcam, Cambridge, MA, USA), mouse anti-β-actin (1:500, Santa, China), mouse anti-c-Fos (1:1000, ab208942, Abcam, Cambridge, MA, USA), rabbit anti-CGRP (1:500, Abcam, Cambridge, MA, USA), mouse anti-TRAF6(1:1,000, SC-7221, China.), rabbit anti-P-TAK1(1:1000, Ser439, MCE, China), mouse anti-TAK1(1:1000, YA663, MCE, China), rabbit anti-P-ERK1/2 (1:1000, 9101S, CST, USA), rabbit anti-ERK1/2 (1:1000, CST, 4695 T, USA), rabbit anti-P-MEK1/2 (1:1000, Ser298, MCE, China), rabbit anti-MEK1/2 (1:1000, ab32576, Abcam, Cambridge, MA, USA).

Techniques: Expressing, Western Blot

Journal: Molecular Pain

Article Title: TLR3 mediates central sensitization in a chronic migraine model induced by repeated nitroglycerin through the ERK signaling pathway

doi: 10.1177/17448069251346373

Figure Lengend Snippet: TLR3 regulates the ERK signaling pathway by TRAF6/TAK1 axis. (a) Immunoblot analysis of p-TAK1 to TAK1 ratio, p-MEK to MEK ratio, p-ERK to ERK ratio, and c-Fos were conducted in the indicated groups ( n = 5). (b) Immunoblot analysis of p-MEK to MEK ratio, p-ERK to ERK ratio, and c-Fos were conducted in the indicated groups ( n = 5). Data are the mean ± SEM; one-way ANOVA and Tukey’s post hoc tests. * p < 0.05, ** p < 0.01, *** p < 0.001 versus the VEH group, and # p < 0.05, ## p < 0.01, ### p < 0.001 versus the NTG group.

Article Snippet: Then, we placed the NC membrane in a TBST solution containing 5% skim milk powder and sealed it at room temperature for 1 h. Next step, we incubated it overnight with the following antibodies at 4°C: mouse anti-TLR3 (1:500, ab13915, Abcam, Cambridge, MA, USA), mouse anti-β-actin (1:500, Santa, China), mouse anti-c-Fos (1:1000, ab208942, Abcam, Cambridge, MA, USA), rabbit anti-CGRP (1:500, Abcam, Cambridge, MA, USA), mouse anti-TRAF6(1:1,000, SC-7221, China.), rabbit anti-P-TAK1(1:1000, Ser439, MCE, China), mouse anti-TAK1(1:1000, YA663, MCE, China), rabbit anti-P-ERK1/2 (1:1000, 9101S, CST, USA), rabbit anti-ERK1/2 (1:1000, CST, 4695 T, USA), rabbit anti-P-MEK1/2 (1:1000, Ser298, MCE, China), rabbit anti-MEK1/2 (1:1000, ab32576, Abcam, Cambridge, MA, USA).

Techniques: Western Blot

Journal: Molecular Pain

Article Title: TLR3 mediates central sensitization in a chronic migraine model induced by repeated nitroglycerin through the ERK signaling pathway

doi: 10.1177/17448069251346373

Figure Lengend Snippet: Blocking TRAF6 and TAK1 alleviates CM-related pain hypersensitivity in CM mice. (a–h) C25-140 and takinib effectively prevented the decrease in both basal and acute mechanical withdrawal thresholds in the periorbital area and the hind paw. Acute hypersensitivity reactions were detected 2 h after each NTG treatment. Data are presented as mean ± SEM; two-way ANOVA and Turkey post hoc analysis; n = 10 per group. ** p < 0.01, *** p < 0.001 compared to the VEH group; # p < 0.05, ## p < 0.01 and ### p < 0.001 compared to the NTG group.

Article Snippet: Then, we placed the NC membrane in a TBST solution containing 5% skim milk powder and sealed it at room temperature for 1 h. Next step, we incubated it overnight with the following antibodies at 4°C: mouse anti-TLR3 (1:500, ab13915, Abcam, Cambridge, MA, USA), mouse anti-β-actin (1:500, Santa, China), mouse anti-c-Fos (1:1000, ab208942, Abcam, Cambridge, MA, USA), rabbit anti-CGRP (1:500, Abcam, Cambridge, MA, USA), mouse anti-TRAF6(1:1,000, SC-7221, China.), rabbit anti-P-TAK1(1:1000, Ser439, MCE, China), mouse anti-TAK1(1:1000, YA663, MCE, China), rabbit anti-P-ERK1/2 (1:1000, 9101S, CST, USA), rabbit anti-ERK1/2 (1:1000, CST, 4695 T, USA), rabbit anti-P-MEK1/2 (1:1000, Ser298, MCE, China), rabbit anti-MEK1/2 (1:1000, ab32576, Abcam, Cambridge, MA, USA).

Techniques: Blocking Assay

Journal: Molecular Pain

Article Title: TLR3 mediates central sensitization in a chronic migraine model induced by repeated nitroglycerin through the ERK signaling pathway

doi: 10.1177/17448069251346373

Figure Lengend Snippet: Schematic diagram of how TLR3 promotes central sensitization by TRAF6-TAK1 axis modulating the ERK signaling pathway.

Article Snippet: Then, we placed the NC membrane in a TBST solution containing 5% skim milk powder and sealed it at room temperature for 1 h. Next step, we incubated it overnight with the following antibodies at 4°C: mouse anti-TLR3 (1:500, ab13915, Abcam, Cambridge, MA, USA), mouse anti-β-actin (1:500, Santa, China), mouse anti-c-Fos (1:1000, ab208942, Abcam, Cambridge, MA, USA), rabbit anti-CGRP (1:500, Abcam, Cambridge, MA, USA), mouse anti-TRAF6(1:1,000, SC-7221, China.), rabbit anti-P-TAK1(1:1000, Ser439, MCE, China), mouse anti-TAK1(1:1000, YA663, MCE, China), rabbit anti-P-ERK1/2 (1:1000, 9101S, CST, USA), rabbit anti-ERK1/2 (1:1000, CST, 4695 T, USA), rabbit anti-P-MEK1/2 (1:1000, Ser298, MCE, China), rabbit anti-MEK1/2 (1:1000, ab32576, Abcam, Cambridge, MA, USA).

Techniques: