murine myd88  (Addgene inc)

Journal: Frontiers in Immunology

Article Title: Fbxo16 mediates degradation of NF-κB p65 subunit and inhibits inflammatory response in dendritic cells

doi: 10.3389/fimmu.2025.1524110

Figure Lengend Snippet: Fbxo16 forms a CRL1 complex, binds to p65 and negatively regulates NF-κB signaling. (A) Interaction of Fbxo16 and Cullins in whole cell lysates of HEK293T cells, transfected with c-Myc-Fbxo16, then immunoprecipitated with anti-Flag, and immunoblotted with anti-Cullin 1, Cullin 2 or Cullin 3. (B) Interaction of Fbxo16 and Skp1 in whole cell lysates of HEK293T cells, transfected with c-Myc-Fbxo16, then immunoprecipitated with anti-c-Myc, and immunoblotted with anti-Skp1. (C) Interaction of Fbxo16 and p65 in whole cell lysates of HEK293T cells, transfected with a Flag-p65 or p50 along without or with c-Myc-Fbxo16, then immunoprecipitated with anti-c-Myc, and immunoblotted with anti-p65 or p50. (D) Interaction of Fbxo16 and PDLIM2 in whole cell lysates of HEK293T cells, transfected with a Flag-PDLIM2 along without or with c-Myc-Fbxo16, then immunoprecipitated with anti-c-Myc, and immunoblotted with anti-Flag. (E) Luciferase assay in MEFs transfected with ELAM−1 luciferase reporter and pRL-Null renilla constructs along with increasing amounts (wedge) of Fbxo16, then left untreated or stimulated with LPS for 5 hr. (F) Luciferase assay in HEK293T cells transfected with ELAM−1 luciferase reporter and pRL-Null renilla constructs without or with MyD88, IKKβ, TRAF6 or p65 along with increasing amounts (wedge) of Fbxo16. Data are representative of three (A, B) , four (C, D) or three ( E, F ; means ± SD, **P<0.01) independent experiments.

Article Snippet: Expression plasmids for Flag-tagged murine p50 (#20018), murine TRAF6 (#21624), murine MyD88 (#13093) and human IKKβ (#23298) were purchased from Addgene.

Techniques: Transfection, Immunoprecipitation, Luciferase, Construct

Journal: BMC complementary medicine and therapies

Article Title: Soyasaponins reduce inflammation by downregulating MyD88 expression and suppressing the recruitments of TLR4 and MyD88 into lipid rafts.

doi: 10.1186/s12906-020-2864-2

Figure Lengend Snippet: Fig. 3 Soyasaponins inhibited the recruitments of TLR4 and MyD88 into lipid rafts in liver tissues. The lipid rafts (fraction 3 and 4) were fractionated from liver tissue lysates by using sucrose gradient ultracentrifugation. The protein levels of flotillin-1 (a lipid raft marker), TLR4 and MyD88 were detected by western blotting. The ratio of the amount of TLR4 (or MyD88) in lipid raft to that in total fractions were quantitatively calculated. Results reported are Means ± SD of samples from six mice in each group (n = 6). Data were statistically analyzed by using one-way ANOVA of SPSS software. *: p < 0.05 v.s. control, #: p < 0.05 v.s. LPS alone

Article Snippet: HEK293T cells were transfected with MyD88 flag expression plasmid (Addgene plasmid #13093) or the empty plasmid vector using the LipofectamineTM 3000 reagent (Invitrogen) by following the recommended protocols.

Techniques: Marker, Western Blot, Software, Control

Journal: BMC complementary medicine and therapies

Article Title: Soyasaponins reduce inflammation by downregulating MyD88 expression and suppressing the recruitments of TLR4 and MyD88 into lipid rafts.

doi: 10.1186/s12906-020-2864-2

Figure Lengend Snippet: Fig. 4 Effects of soyasaponins on the protein levels of molecules in TLR4/MyD88 signaling pathway in LPS-stimulated RAW264.7 macrophages. RAW264.7 macrophages were pre-treated with graded concentrations (10, 20 or 40 μmol/L) of soyasaponins (A1, A2, or I) for 2 h and then stimulated with LPS (1 μg/mL) for 30 min (a and b), 1 h (c), or 3 h (d and e). The levels of molecules (MD-2, TLR4, TIRAP, MyD88, p-IRAK4, p-IRAK1 and TRAF6) in TLR4/MyD88 signaling pathway were measured by western blotting. Results reported are Means ± SD of three independent experiments. Data were statistically analyzed by using one-way ANOVA of SPSS software. *: p < 0.05 v.s. control, #: p < 0.05 v.s. LPS alone

Article Snippet: HEK293T cells were transfected with MyD88 flag expression plasmid (Addgene plasmid #13093) or the empty plasmid vector using the LipofectamineTM 3000 reagent (Invitrogen) by following the recommended protocols.

Techniques: Western Blot, Software, Control

Journal: BMC complementary medicine and therapies

Article Title: Soyasaponins reduce inflammation by downregulating MyD88 expression and suppressing the recruitments of TLR4 and MyD88 into lipid rafts.

doi: 10.1186/s12906-020-2864-2

Figure Lengend Snippet: Fig. 5 Soyasaponins inhibit the protein expression of MyD88 and TRAF6, and activation of NF-κB in MyD88-transfected HEK293T cells. HEK293T cells were transfected with MyD88-flag plasmid for 24 h, and then treated with graded concentration (20 or 40 μmol/L) of soyasaponin (A1, A2 or I) or ST2825 (a MyD88 inhibitor) for 6 h. The protein levels of TLR4 (a), MyD88 (b), TRAF6 (c), p-p65 and p65 (d) were measured by western blotting. Results reported are Means ± SD of three independent experiments. Data were statistically analyzed by using one-way ANOVA of SPSS software. *: p < 0.05 v.s. control, #: p < 0.05 v.s. MyD88-flag plasmid transfected group

Article Snippet: HEK293T cells were transfected with MyD88 flag expression plasmid (Addgene plasmid #13093) or the empty plasmid vector using the LipofectamineTM 3000 reagent (Invitrogen) by following the recommended protocols.

Techniques: Expressing, Activation Assay, Transfection, Plasmid Preparation, Concentration Assay, Western Blot, Software, Control

Journal: BMC complementary medicine and therapies

Article Title: Soyasaponins reduce inflammation by downregulating MyD88 expression and suppressing the recruitments of TLR4 and MyD88 into lipid rafts.

doi: 10.1186/s12906-020-2864-2

Figure Lengend Snippet: Fig. 5 Soyasaponins inhibit the protein expression of MyD88 and TRAF6, and activation of NF-κB in MyD88-transfected HEK293T cells. HEK293T cells were transfected with MyD88-flag plasmid for 24 h, and then treated with graded concentration (20 or 40 μmol/L) of soyasaponin (A1, A2 or I) or ST2825 (a MyD88 inhibitor) for 6 h. The protein levels of TLR4 (a), MyD88 (b), TRAF6 (c), p-p65 and p65 (d) were measured by western blotting. Results reported are Means ± SD of three independent experiments. Data were statistically analyzed by using one-way ANOVA of SPSS software. *: p < 0.05 v.s. control, #: p < 0.05 v.s. MyD88-flag plasmid transfected group

Article Snippet: HEK293T cells were transfected with MyD88 flag expression plasmid (Addgene plasmid #13093) or the empty plasmid vector using the LipofectamineTM 3000 reagent (Invitrogen) by following the recommended protocols.

Techniques: Expressing, Activation Assay, Transfection, Plasmid Preparation, Concentration Assay, Western Blot, Software, Control