mouse anti tak1  (MedChemExpress)

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:

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Influenza A virus infection activates caspase-8 to enhance innate antiviral immunity by cleaving CYLD and blocking TAK1 and RIG-I deubiquitination

doi: 10.1007/s00018-024-05392-z

Figure Lengend Snippet: Caspase-8 enhances RIG-I and TAK1 ubiquitination by inactivating CYLD. ( A-D ) A549 cells were cotransfected with the expression vector encoding the HA-ubiquitin gene plus the FLAG-RIG-I gene ( A ) or the FLAG-TAK1 gene ( C ). pLenti-V2-transfected WT control and caspase-8- or CYLD-deficient A549 cells were cotransfected with the expression vector encoding the HA-ubiquitin gene plus the FLAG-RIG-I gene ( B ) or the FLAG-TAK1 gene ( D ). After incubation for 24 h, the cells were left uninfected or infected with H5N1 (SY) virus (1 MOI) and then incubated for another 24 h. Cell lysates were prepared and immunoprecipitated with an antibody against FLAG. Whole-cell lysates (WCLs) and immunoprecipitates were analyzed with antibodies against FLAG and HA. The data represent one of three independent experiments. ( E ) Schematic model of caspase-8-mediated immune activation. Influenza A virus activates TBK1 and TAK1 through RIG-I and TRL3, which then activate IRF3 and NF-κB. IAV infection activates caspase-8 in a ZBP1-dependent (NL20 cells) and ZBP1-independent (A549 cells) manner. Activated caspase-8 cleaves and inactivates CYLD, a deubiquitinase that removes the K63 sidechains of RIG-I and TAK1. Increased RIG-I and TAK1 ubiquitination enhances the activation of the TBK1-IRF3 and TAK1-NF-κB pathways, leading to strong antiviral responses such as increased IFN and ISG gene expression and the restriction of virus replication

Article Snippet: Antibodies against cleaved caspase-8 (#9496), cleaved caspase-8 (#4790), cleaved caspase-3 (#9664), cleaved caspase-3 (#14220), CYLD (#8462), phospho-p65 (#3033), p65 (#3308), phospho-IκBα (#9341 s), IκBα (#9242), phospho-TBK1 S172 (#5483), TBK1 (#3504), phospho-IRF3 S396 (#29047), IRF3 (#4302), RIG-I (#3743), RIPK1 (#sc3493), phospho-TAK1 S187 (#4536), TAK1 (#4505), JAK1 (#50996), and HA tag (#2367) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).

Techniques: Ubiquitin Proteomics, Expressing, Plasmid Preparation, Transfection, Control, Incubation, Infection, Virus, Immunoprecipitation, Activation Assay, Gene Expression