recombinant human myd88  (Novus Biologicals)

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Schisandrin B Attenuates Diabetic Cardiomyopathy by Targeting MyD88 and Inhibiting MyD88-Dependent Inflammation.

doi: 10.1002/advs.202202590

Figure Lengend Snippet: Figure 3. Sch B exhibits differential regulation of TLR4/TRIF and TLR4/MyD88 pathways. A) Immunofluorescence staining of H9C2 cells for p65 subunit of NF-𝜅B (red). Cells were pretreated with 10 × 10−6 m Sch B for 1 h and then exposed to HG for 1 h. Cells were counterstained with DAPI (blue) (scale bar = 100 μm). B) Immunoblot analysis of I𝜅B𝛼in total cell lysates and p65 in nuclear fractions prepared from H9C2 cells. Cells were treated as indicated for panel (A). GAPDH was used as control for total cell lysates and lamin B for nuclear proteins. C) Western blot analysis of MAPK activation in H9C2 cells. Cells were treated as in panel (B). Total cell lysates were probed for phosphorylated (p-) and total ERK, JNK, and p38. D) Levels of MyD88 pathway protein TAK1 in H9C2 cells exposed to HG. Cells were treated as indicated for panel (B). Upper blots showing H9C2 cells and lower blots showing primary cardiomyocytes. Densitometric quantification of blots is shown in right panel. E) Analysis of TRIF pathway showing levels of TBK1 and IRF3. H9C2 (upper blots) and primary cardiomyocytes (lower blots) were treated as indicated for panel (B). Densitometric quantification is shown on right. F) Co- immunoprecipitation analysis to examine the MD2/TLR4, TLR4/TRIF, and TLR4/MyD88 complexes in H9C2 cells. Cells were pretreated with 10 × 10−6 m Sch B for 1 h and then exposed to HG for 30 min. TLR4 was immunoprecipitated (IP) and TRIF and MyD88 were detected by immunoblotting (IB). In lower blots, MD2 was immunoprecipitated (IP) and TLR4 was detected by immunoblotting (IB). G) Co-immunoprecipitation of MD2/TLR4, TLR4/TRIF, and TLR4/MyD88 complexes in primary cardiomyocytes. Cells were treated as indicated for panel (F). Densitometric quantification is shown in the lower panel. All data in panels (D)–(G) is shown as mean ± SEM (n = 3; *p < 0.05, **p < 0.01 compared to Ctrl and ##p < 0.01, ns = not significant compared to HG by one-way ANOVA followed by Bonferroni’s multiple comparisons test).

Article Snippet: Recombinant human MyD88 (rhMyD88) was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Staining, Western Blot, Control, Activation Assay, Immunoprecipitation

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Schisandrin B Attenuates Diabetic Cardiomyopathy by Targeting MyD88 and Inhibiting MyD88-Dependent Inflammation.

doi: 10.1002/advs.202202590

Figure Lengend Snippet: Figure 4. Sch B directly binds to MyD88. A) Western blot analysis of MyD88 knockdown in H9C2 cells. Cells were transfected with negative control siRNA (NC) or siRNA against MyD88 (siMyD88). GAPDH was used as loading control. (Values represent the mean ± SEM; n = 3; ***p < 0.001 compared to NC by unpaired two-tailed Student’s t-test). B) qPCR analysis of inflammatory cytokines in H9C2 cells transfected with MyD88 siRNA. Transfected cells were treated with 10 × 10−6 m Sch B for 1 h before exposure to HG for 8 h. Untransfected (Ctrl) and cells transfected with negative control siRNA (NC) were used as control (values represent mean ± SEM; n = 3; ns = not significant; *p < 0.05; ***p < 0.001 compared to control, and #p < 0.05 compared to HG+siNC by unpaired two-tailed Student’s t-test). C) H9C2 cells were transfected with Flag- and HA-tagged MyD88. Cells were treated with 10 × 10−6 m Sch B for 1 h and then exposed to HG for 30 min. Flag was immunoprecipitated and HA was detected to examine MyD88 dimerization. D) Binding of biotinylated-Sch B to MyD88 was determined by immunoblotting. Bio-Sch B was added to streptavidin-agarose beads. Untreated beads, unconjugated Sch B, and biotin alone were used as control. Lysates prepared from control mouse heart tissues were added. E) Western blot analysis of the binding of Bio-Sch B to MyD88-TIR domain. Lysates prepared from HEK 293T cells transfected with Flag-tagged full MyD88 or Flag-tagged MyD88 TIR domain only were incubated with Bio-Sch B-loaded beads or biotin-loaded beads. Upper panel shows the structure of MyD88 and lower panel shows pulled proteins. F) Fluorescence spectroscopy utilizing bis-ANS showing the binding of Sch B to rhMyD88. G) SPR analysis showing interaction between Sch B and recombinant MyD88 protein. Sch B was added at different concentrations and KD values were calculated (shown in the insert). H) ITC analysis for MyD88 binding to Sch B. Representative image shown. The left panel shows the representative titration thermograms, and the right panel shows the data integration with fitted curves (independent model) of Sch B with MyD88(TIR). I) The binding pocket of Sch B and the key residues with the lowest binding energy. Left panel shows the carbon atoms of six key residues’ side chain and Sch B represented as green sticks and yellow sticks, respectively. Right panel shows the boxplot of the per-residue decomposition energy of the six residues. Red arrows indicate potentially important residues. J) Pull- down analysis of the binding of Bio-Sch B to mutant MyD88 containing T272A and R288A. HEK 393T cells were transfected with wildtype MyD88 or mutant variants. Lysates were used to detect binding to bio-Sch B (mean ± SEM; n = 3; ns = not significant; *p < 0.05 compared to Bio; #p < 0.05 compared to Bio-Sch B by unpaired two-tailed Student’s t-test).

Article Snippet: Recombinant human MyD88 (rhMyD88) was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Western Blot, Knockdown, Transfection, Negative Control, Control, Two Tailed Test, Immunoprecipitation, Binding Assay, Incubation, Fluorescence, Spectroscopy, Recombinant, Titration, Residue, Mutagenesis

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Schisandrin B Attenuates Diabetic Cardiomyopathy by Targeting MyD88 and Inhibiting MyD88-Dependent Inflammation.

doi: 10.1002/advs.202202590

Figure Lengend Snippet: Figure 6. Sch B reduces inflammatory responses in db/db mice by inhibiting MyD88. A) Co-immunoprecipitation showing formation of MD2/TLR4, TLR4/MyD88, and TLR4/TRIF complexes in heart tissues of db/db mice. TLR4 was immunoprecipitated (IP) and levels of MD2, TRIF, and MyD88 were detected by immunoblotting (IB). Densitometric quantification is shown on right. B) Western blot analysis of pathway activation downstream of MyD88 in heart tissues. Phosphorylated TAK1, TBK1, and IRF3 were detected. Total proteins were used as control. Densitometric quantification shown on right. C) Analysis of MAPK and NF-𝜅B activation in heart tissues of db/db mice. Phosphorylated ERK, JNK, and p38 were detected. Total proteins were used as control. I𝜅B-𝛼was used to measure NF-𝜅B activity, with GAPDH as the loading control. Right panel shows densitometric quantification. D) mRNA levels of Tnf, Il6, and Ifng in heart tissues of mice. Transcripts were normalized to Actb. E) Protein levels of IL-6 and TNF-𝛼in serum of db/db mice were determined by enzyme-linked immunoassay (ELISA). Data in all panels are shown as mean ± SEM (n = 6 per group; *p < 0.05, **p < 0.01, ***p < 0.001 compared to db/m; #p < 0.05, ##p < 0.01, ns = not significant compared to db/db by one-way ANOVA followed by Bonferroni’s multiple comparisons test).

Article Snippet: Recombinant human MyD88 (rhMyD88) was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Immunoprecipitation, Western Blot, Activation Assay, Control, Activity Assay, Enzyme-linked Immunosorbent Assay

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Schisandrin B Attenuates Diabetic Cardiomyopathy by Targeting MyD88 and Inhibiting MyD88-Dependent Inflammation.

doi: 10.1002/advs.202202590

Figure Lengend Snippet: Figure 7. Sch B treatment suppresses cardiac injury in STZ-induced model of type 1 diabetes. A,B) Representative images of A) Masson’s trichrome and B) Sirius red staining of heart tissues (scale bar = 50 μm). C) mRNA levels of Cola1, Anp, and Ska in heart tissues. Transcripts were normalized to Actb. D) TLR4 was immunoprecipitated (IP) from mouse heart lysates and MD2, TRIF, and MyD88 were detected by immunoblotting (IB). Right panel

Article Snippet: Recombinant human MyD88 (rhMyD88) was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Staining, Immunoprecipitation, Western Blot

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Schisandrin B Attenuates Diabetic Cardiomyopathy by Targeting MyD88 and Inhibiting MyD88-Dependent Inflammation.

doi: 10.1002/advs.202202590

Figure Lengend Snippet: Figure 8. Cardiomyocyte-specific MyD88 knockout prevents STZ-induced cardiac inflammation and injury. Male MyD88f/f and MyD88f/fMyh6Cre mice were injected with STZ to induce T1DM. A) Fasting blood glucose and B) body weight were recorded weekly for 16 weeks. Heart tissue and blood samples were collected at termination for the determination of C) heart weight/tibia length (HW/TL) ratios, D) serum LDH, and E) serum CK-MB

Article Snippet: Recombinant human MyD88 (rhMyD88) was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Knock-Out, Injection

Journal: Nature communications

Article Title: Orphan receptor IL-17RD regulates Toll-like receptor signalling via SEFIR/TIR interactions.

doi: 10.1038/ncomms7669

Figure Lengend Snippet: Figure 1 | IL-17RD negatively regulates TLR signalling pathways. (a,b) Assay of NF-kB-regulated luciferase reporter activity in HEK293 cells transfected with Myc-tagged IL-17RD (0–100 ng) and (a) MyD88, Mal, TRIF or TRAM (50 ng) with a NF-kB luciferase reporter plasmid (60 ng) or (b) with TRIF (50 ng) and PFR-luciferase (60 ng) with IRF3-Gal4 (30 ng) or IRF7-Gal4 (25 ng) constructs. TK Renilla was measured to determine transfection efficiency. (c,d) Supernatants were measured for (c) IL-8 or (d) RANTES production by sandwich ELISA. (e,f) ELISA of (e) TNF-a or (f) IL-8 from U373 or THP-1 cells, respectively, previously transduced with lentiviral-encoded control or IL-17RD-specific shRNA and treated with LPS (100 ng ml 1) for 24 h. (g) Supernatants from U373 cells stably transduced with control or IL-17RD-specific shRNA measured for RANTES and IP-10 levels by ELISA in response to poly(I:C) (25 mg ml 1). (h,i) Cell lysates from (h) THP-1 cells treated with LPS (100 ng ml 1) for 0, 5, 15, 30, 60 and 240 min and (i) U373 cells treated with poly(I:C) (25 mg ml 1) for 0, 45, 90, 120, 240 and 360 min, both stably expressing control and IL-17RD-specific shRNA, were subjected to immunoblotting with indicated antibodies. (h,i: inset panels) Cell lysates from THP-1 and U373 cells expressing control (Ctrl) or IL-17RD-specific shRNA (shRNA) were analysed for IL-17RD and b-actin expression with specific antibodies by immunoblotting. Overexpressed IL-17RD (oe) was used as a control from HEK293 and U373 cell lysates, respectively. Data are presented (a–g) as the mean ±s.e.m. of three independent experiments and were subjected to a two-tailed paired Student’s t-test, *Po0.05; **Po0.01 or (h,i) are representative of three independent experiments. Images have been cropped for presentation. Full-size images are presented in Supplementary Fig. 7.

Article Snippet: Recombinant human MyD88 was from Novus Biologicals (Littleton, CO) and recombinant human IL-17RD was from Origene (Rockville, MD).

Techniques: Luciferase, Activity Assay, Transfection, Plasmid Preparation, Construct, Sandwich ELISA, Enzyme-linked Immunosorbent Assay, Transduction, Control, shRNA, Stable Transfection, Expressing, Western Blot, Two Tailed Test

Journal: Nature communications

Article Title: Orphan receptor IL-17RD regulates Toll-like receptor signalling via SEFIR/TIR interactions.

doi: 10.1038/ncomms7669

Figure Lengend Snippet: Figure 4 | SEFIR domain is critical for inhibitory effects of IL-17RD. (a) HEK293 cells were transfected for 24 h with (a) MyD88-FLAG, Mal-FLAG and IL-17RD-Myc (1 mg). Cell lysates were immunoprecipitated with an anti-FLAG antibody, followed by immunoblotting with indicated antibodies. (b) HEK293 cells were transfected for 24 h with MyD88-GFP and IL-17RD-RFP and visualized for expression and localization using confocal microscopy. Cells were also incubated with nuclei-staining DAPI. Confocal images were captured using the 63 objective (oil immersion) on the UV Zeiss 510 Meta System laser scanning microscope equipped with the appropriate filter sets and analysed using the LSM 5 browser imaging software (scale bar, 20 mm). (c) HEK293 cells were transfected for 24 h with MyD88-FLAG with IL-17RD-Myc or IL-17RD DC-Myc (1 mg). Cell lysates were immunoprecipitated with an anti-FLAG antibody, followed by immunoblotting with indicated antibodies. (d) Assay of NF-kB-regulated luciferase expression in HEK293 cells transfected with MyD88 (50 ng) with IL-17RD-Myc or IL-17RD DC-Myc (50 ng). (e–g) HEK293 cells were transfected for 24 h with (e) TLR4-FLAG and IL-17RD-Myc or IL-17RD DC-Myc, (f) MyD88-FLAG and IL-17RD-Myc, IL-17RD DC-Myc and IL-17RD SEFIR-Myc or (g) TLR4-FLAG and IL-17RD SEFIR-Myc (1 mg). Cell lysates were immunoprecipitated with an anti-FLAG antibody, followed by immunoblotting with indicated antibodies. (h) Assay of NF-kB regulated luciferase expression in HEK293 cells transfected with MyD88 (50 ng) and IL-17RD-Myc or IL-17RD SEFIR-Myc (100 ng). Data are (a–c,e–g) representative of three independent experiments or (d,h) are presented as the mean ±s.e.m. of three independent experiments and were subjected to a two-tailed paired Student’s t-test. *Po0.05. Images have been cropped for presentation. Full-size images are presented in Supplementary Fig. 9. NS, not significant.

Article Snippet: Recombinant human MyD88 was from Novus Biologicals (Littleton, CO) and recombinant human IL-17RD was from Origene (Rockville, MD).

Techniques: Transfection, Immunoprecipitation, Western Blot, Expressing, Confocal Microscopy, Incubation, Staining, Laser-Scanning Microscopy, Imaging, Software, Luciferase, Two Tailed Test

Journal: Nature communications

Article Title: Orphan receptor IL-17RD regulates Toll-like receptor signalling via SEFIR/TIR interactions.

doi: 10.1038/ncomms7669

Figure Lengend Snippet: Figure 5 | IL-17RD disrupts signalling downstream of TIR adaptors. (a) WT and Il17rd / BMDMs were stimulated for 0, 5, 20 and 60 min with LPS (10 ng ml 1) and cell lysates were subject to immunoprecipitation with an anti-MyD88 antibody and subjected to immunoblotting with indicated antibodies. (b,c) WT and Il17rd / BMDMs or BMDCs were treated for (b) 0, 5, 30 and 60 min with LPS (10 ng ml 1) or (c) 0, 30, 90 and 180 min poly(I:C) (5 mg ml 1), respectively. Cell lysates were subject to SDS treatment, immunoprecipitation with an anti-TRAF6 antibody and probed by immunoblotting with indicated antibodies. (d,e,g) HEK293 cells were transfected for 24 h with MyD88-FLAG with (d) TRAF6-HA with IL-17RD-Myc, IL-17RD DC-Myc or IL-17RD SEFIR-Myc (total 3 mg) or (e) Myc-tagged IL-17RD SEFIR, IL-17RD SEFIR DBox1 or IL-17RD SEFIR DBox3 or (g) Myc-tagged IL-17RD SEFIR, IL-17RD SEFIR T496P, IL-17RD SEFIR K497R, IL-17RD SEFIR L504F or IL-17RD SEFIR T496P/K497R. Cell lysates were immunoprecipitated with an anti-FLAG antibody, followed by immunoblotting with indicated antibodies. (f,h) Assay of NF-kB-regulated luciferase expression in HEK 293 cells transfected with MyD88 (50 ng) with (f) Myc-tagged IL-17RD SEFIR, IL-17RD SEFIR DBox1 or IL-17RD SEFIR DBox3 (100 ng) or (h) Myc-tagged IL-17RD SEFIR, IL-17RD SEFIR T496P, IL-17RD SEFIR K497R, IL-17RD SEFIR L504F or IL-17RD SEFIR T496P/K497R (100 ng). Data are (a–e,g) representative of three independent experiments or (f,h) are presented as the mean ±s.e.m. of three independent experiments and were subjected to a two-tailed paired Student’s t-test. *Po0.05; **Po0.01. Images have been cropped for presentation. Full-size images are presented in Supplementary Fig. 10. NS, not significant.

Article Snippet: Recombinant human MyD88 was from Novus Biologicals (Littleton, CO) and recombinant human IL-17RD was from Origene (Rockville, MD).

Techniques: Immunoprecipitation, Western Blot, Transfection, Luciferase, Expressing, Two Tailed Test

Journal: Nature communications

Article Title: Orphan receptor IL-17RD regulates Toll-like receptor signalling via SEFIR/TIR interactions.

doi: 10.1038/ncomms7669

Figure Lengend Snippet: Figure 6 | LPS induces in situ association of IL-17RD with MyD88. (a) PBMCs from human donor blood were treated with LPS (100 ng ml 1) for 0, 1, 5, 20 and 60 min and cell lysates were subject to immunoprecipitation with an anti-IL-17RD antibody. Cell lysate and immunoprecipitate samples were immunoblotted with indicated antibodies. (b) PBMCs from human donor blood were transfected with scrambled or IL-17RD-specific siRNA and cultured for 48 h prior to treatment in the absence or presence of LPS (100 ng ml 1) for 20 min. Cells were fixed to slides and subjected to Duolink in situ proximity ligation assay using anti-IL-17RD and anti-MyD88 antibodies. IL-17RD/MyD88 interactions are visible as red fluorescence of the Duolink detection reagents with nuclear staining in blue (DAPI). Images were captured using the 20 objective of a fluorescent microscope with scale bars representing 50 mm. The histogram represents the area of red Duolink signal as a percentage of area of blue DAPI signal from five random images from each of three independent experiments. Probe alone indicates areas of signals from cells subjected to ligation assay, but in the absence of anti-IL-17RD and anti-MyD88 antibodies (five random images from each of the two independent experiments). Data are presented as the mean ±s.e.m. and were subjected to a two-tailed paired Student’s t-test. *Po0.05; **Po0.01. The lowest-right panel demonstrates knockdown of IL-17RD expression by IL-17RD-specific siRNA as determined by immunoblotting of cell lysates from cells treated with LPS with or without prior transfection with IL-17RD-specific siRNA or a scrambled sequence version of this siRNA. Images have been cropped for presentation. Full-size images are presented in Supplementary Fig. 11.

Article Snippet: Recombinant human MyD88 was from Novus Biologicals (Littleton, CO) and recombinant human IL-17RD was from Origene (Rockville, MD).

Techniques: In Situ, Immunoprecipitation, Transfection, Cell Culture, Proximity Ligation Assay, Staining, Microscopy, Ligation, Two Tailed Test, Knockdown, Expressing, Western Blot, Sequencing