Journal: International immunology

Article Title: A variety of microbial components induce tolerance to lipopolysaccharide by differentially affecting MyD88-dependent and -independent pathways.

doi: 10.1093/intimm/dxf046

Figure Lengend Snippet: Fig. 6. Expression of IRAK1 was severely reduced in R-848-pretreated macrophages. (A) Macrophages were treated with the indicated stimuli for 24 h. Whole-cell lysates were prepared and subjected to Western blot analysis using antibodies for MyD88, IRAK1, TRAF6 and Tollip. (B) Cell lysates were immunoprecipitated with anti-IRAK1 antibody. The kinase activity of IRAK1 in the immunoprecipitates was measured by in vitro kinase assay (upper panel). The same lysates were blotted with anti-IRAK1 antibody to monitor the expression level of IRAK1 (lower panel). The results are representative of three independent experiments that had similar results. L, LPS; R, R-848; P, poly(I:C).

Article Snippet: Rabbit anti-MyD88 antibody was purchased from ProSci (Poway, CA).

Techniques: Expressing, Western Blot, Immunoprecipitation, Activity Assay, In Vitro, Kinase Assay

Journal: International immunology

Article Title: A variety of microbial components induce tolerance to lipopolysaccharide by differentially affecting MyD88-dependent and -independent pathways.

doi: 10.1093/intimm/dxf046

Figure Lengend Snippet: Fig. 7. Pretreatment with poly(I:C), but not R-848, led to the impaired LPS-induced expression of IFN-inducible genes. (A and B) Peritoneal macrophages were prepared from wild-type (A) or MyD88±/± (B) mice, and then pretreated with the indicated stimuli for 24 h. Cells were washed twice and stimulated with LPS for 4 h. Total RNA was extracted and subjected to Northern blot analysis using cDNA probes for the indicated genes. (C) Peritoneal macrophages were pretreated with poly(I:C) or LPS for 24 h. Cells were washed twice and then stimulated with LPS for the indicated periods. Cell lysates were prepared and subjected to native PAGE. Monomeric (arrow) and dimeric (arrowhead) forms of IRF-3 were detected by Western blotting. The results are representative of three independent experiments that had similar results. L, LPS; R, R-848; P, poly(I:C).

Article Snippet: Rabbit anti-MyD88 antibody was purchased from ProSci (Poway, CA).

Techniques: Expressing, Northern Blot, Clear Native PAGE, Western Blot

Journal: Genes

Article Title: RNAi-Induced Expression of Paternal UBE3A

doi: 10.3390/genes17020156

Figure Lengend Snippet: Activation of paternal Ube3a by inhibiting Ube3a-ATS using siRNA targeting the Snord115 region in mouse primary neurons. ( a ) Schematic diagram of the paternal allele in the Ube3a P-YFP/m+ mouse model. ( b ) Representative immunofluorescence staining for YFP (paternal Ube3a) following the indicated treatment (ASOs; 5 μM, siRNA; 5 nM). The red signal shows YFP expression. ( c ) Gene expression of Snrpn , Snord116 , Snord115 , Ube3a , and Ube3a-ATS in Ube3a P-YFP/m+ primary neurons following each treatment after 72 h. Each gene expression level was normalized by the control. Topotecan (300 nM) was normalized with Mock, ASO A was normalized with ASO control, and si Snord115 was normalized with siUNC. Each bar represents the mean value from five technical replicate wells, and the results were confirmed in at least two independent experiments. The graph shown is a representative dataset from one independent experiment. ( d ) Representative image of the Ube3a protein in Ube3a P-YFP/m+ primary neurons following each treatment. The numbers below each band indicate the fold-change in band intensity relative to the corresponding control, normalized to tubulin. Western blotting was performed independently at least two times, and representative results are shown. ( e ) Schematic diagram of the paternal allele in the Ube3a p+/m+ mouse model. ( f ) Gene expression of Snrpn , Snord116 , Snord115 , Ube3a , and Ube3a-ATS in Ube3a p+/m− primary neurons following each treatment after 72 h. Each gene expression level was normalized by the control. Topotecan (300 nM) was normalized with Mock, ASO A was normalized with ASO control, and siSnord115 was normalized with siUNC. Each bar represents the mean value from six technical replicate wells, and the results were confirmed in at least two independent experiments. The graph shown is a representative dataset from one independent experiment. ( g ) Representative image of the Ube3a protein in Ube3a P-YFP/m+ primary neurons following each treatment. The numbers below each band indicate the fold-change in band intensity relative to the corresponding control, normalized to tubulin. Western blotting was performed independently at least two times, and representative results are shown. All data in ( c , f ) are presented as mean ± SEM. Two-way ANOVA with repeated measures was used for significance analyses. Dotted lines are included as visual guides to facilitate interpretation of the data. n.s.; not significant * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 compared with each control. The white scale bar represents 50 μm.

Article Snippet: Following incubation, the membranes were washed three times with Tris-buffered saline with 0.1% TWEEN 20 (TBS-T) and subsequently incubated with the appropriate secondary antibody for 1 h at RT: Anti-GFP (Millipore-Sigma, 06-896, USA), anti-UBE3A (Sigma, SAB1404508, USA), hFABTM Rhodamine Anti-Tubulin antibody (Biorad, 12004165, Hercules, CA, USA), Goat Anti-Mouse IgG StarBright Blue 700 (Biorad, 12004158, Hercules, CA, USA), or Goat Anti-Rabbit IgG StarBright Blue 700 (Biorad, 12004161, Hercules, CA, USA).

Techniques: Activation Assay, Immunofluorescence, Staining, Expressing, Gene Expression, Control, Western Blot

Journal: Genes

Article Title: RNAi-Induced Expression of Paternal UBE3A

doi: 10.3390/genes17020156

Figure Lengend Snippet: Activation of paternal Ube3a by inhibiting Ube3a-ATS upon LV-sh Snord115 transduction in Ube3a p + /m− mouse primary neurons. ( a ) Gene expression (72 h after transduction) of Snrpn , Snord116 , Snord115 , Ube3a , and Ube3a-ATS in Ube3a p+/m− primary neurons following transduction with LV-shSnord115, normalized with LV-shscramble at different MOIs. Relative gene expression levels were normalized to eif4a2 expression and expressed relative to the control condition. This graph is representative of data obtained from four independent wells per condition. We repeated at least two independent experiments. Data present the mean ± SEM. Two-way ANOVA with repeated measures was used for significance analyses. **** p < 0.0001 ( Ube3a ) and †††† p < 0.0001 ( Ube3a-ats ) compared with LV-scramble. The Half Maximal Inhibitory Concentration (IC50) was calculated with nonlinear regression (curve fit), log (inhibitor) vs. response variable slope (four parameters) by using Prism software. Dotted lines are included as visual guides to facilitate interpretation of the data ( b ) Ube3a protein expression in Ube3a p+/m− primary neurons following transduction with LV-shscramble and LV-shSnord115 at 72 h at an MOI of 0.5 and 2.0, analyzed by Western blotting. Topotecan (300 nM) was used as a positive control. Western blotting data are shown as a representative figure. ( c ) Relative Ube3a band intensity. The Ube3a protein is normalized with Tubulin and re-normalized with WT-mock. Data present the mean ± SEM of 4 technical replicates from two biological replicates. An ordinary one-way ANOVA with repeated measures was used for significance analyses. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 compared with each group. ( d ) Representative immunofluorescence staining for Ube3a following the indicated treatment. The red signal shows Ube3a expression. The white scale bar represents 50 μm.

Article Snippet: Following incubation, the membranes were washed three times with Tris-buffered saline with 0.1% TWEEN 20 (TBS-T) and subsequently incubated with the appropriate secondary antibody for 1 h at RT: Anti-GFP (Millipore-Sigma, 06-896, USA), anti-UBE3A (Sigma, SAB1404508, USA), hFABTM Rhodamine Anti-Tubulin antibody (Biorad, 12004165, Hercules, CA, USA), Goat Anti-Mouse IgG StarBright Blue 700 (Biorad, 12004158, Hercules, CA, USA), or Goat Anti-Rabbit IgG StarBright Blue 700 (Biorad, 12004161, Hercules, CA, USA).

Techniques: Activation Assay, Transduction, Gene Expression, Expressing, Control, Concentration Assay, Software, Western Blot, Positive Control, Immunofluorescence, Staining

Journal: Genes

Article Title: RNAi-Induced Expression of Paternal UBE3A

doi: 10.3390/genes17020156

Figure Lengend Snippet: Activation of paternal Ube3a by inhibiting Ube3a-ATS upon LV-shSNORD115 transduction in healthy iPSC-derived neurons. ( a ) Schematic diagram of the siRNA screening experiment in SH-SY5Y cells with RA treatment. siRNAs were transfected at 6 days after RA treatment, and the cells were harvested at 24 h after transfection. ( b ) Gene expression of UBE3A and UBE3A-ATS in SH-SY5Y with RA treatment. Each value is an average value from 3 independent experiments. ( c ) LV-sh SNORD115 #1 and #2 were transduced to healthy human iPSC-derived neurons at 8 days after the maturation step started, and the cells were harvested 3 days post-transduction. ( d , e ) Gene expression (72 h after transduction) of UBE3A and UBE3A-ATS in healthy human iPSC-derived neurons following transduction with LV-shSNORD115 #1 and #2, normalized with LV-shscramble at different MOIs. Each data point represents the mean value from two independent experiments. Red dotted line: UBE3A by Topotecan (3 μM); blue dotted line: UBE3A-ATS by Topotecan (3 μM). ( f ) Representative figure of human iPSC-derived neurons. GFP (Green) indicates LV-derived GFP expression. Β-tubulin III (Red) is a marker of matured neurons. Nuclei are visualized by DAPI (Blue) ( d , e ) are presented as mean ± SEM. Two-way ANOVA with repeated measures was used for significance analyses. Dotted lines are included as visual guides to facilitate interpretation of the data ** p < 0.01, *** p < 0.001 and **** p < 0.0001, compared with shscramble at each MOI. The white scale bar represents 100 μm.

Article Snippet: Following incubation, the membranes were washed three times with Tris-buffered saline with 0.1% TWEEN 20 (TBS-T) and subsequently incubated with the appropriate secondary antibody for 1 h at RT: Anti-GFP (Millipore-Sigma, 06-896, USA), anti-UBE3A (Sigma, SAB1404508, USA), hFABTM Rhodamine Anti-Tubulin antibody (Biorad, 12004165, Hercules, CA, USA), Goat Anti-Mouse IgG StarBright Blue 700 (Biorad, 12004158, Hercules, CA, USA), or Goat Anti-Rabbit IgG StarBright Blue 700 (Biorad, 12004161, Hercules, CA, USA).

Techniques: Activation Assay, Transduction, Derivative Assay, Transfection, Gene Expression, Expressing, Marker

Journal: International journal of immunopathology and pharmacology

Article Title: Corilagin relieves atherosclerosis via the toll-like receptor 4 signaling pathway in vascular smooth muscle cells.

doi: 10.1177/03946320241254083

Figure Lengend Snippet: Figure 2. Effect of ox-LDL on the TLR4 signaling pathway in MOVAS cells. (a) mRNA expression of TLR4, TIRAP and MyD88 was measured by qRT-PCR. *p < .05 compared with other concentrations of ox-LDL groups or control group as determined by one-way ANOVA and subsequent Student–Newman–Keuls q-test (n = 5). (b) *p < .05 compared with either time point or control groups determined by one-way ANOVA and subsequent Student–Newman–Keuls q-test (n = 5).

Article Snippet: Afterwards, the cells were subjected to a 2-h incubation at 37°C with either primary anti-TLR4 antibody (1:200 dilution; Cat No. BA1717; Boster Biological Technology, Pleasanton, CA, USA) or anti-MyD88 antibody (1:400 dilution; Cat No. PB9148; Boster Biological Technology, Pleasanton, CA, USA).

Techniques: Expressing, Quantitative RT-PCR, Control

Journal: International journal of immunopathology and pharmacology

Article Title: Corilagin relieves atherosclerosis via the toll-like receptor 4 signaling pathway in vascular smooth muscle cells.

doi: 10.1177/03946320241254083

Figure Lengend Snippet: Figure 3. Effect of corilagin on the TLR4 signaling pathway in MOVAS cells stimulated by ox-LDL. (a) mRNA expression of TLR4, TIRAP, MyD88, TRAF6, p38, NEMO and IRF5 was measured by qRT-PCR. *p < .05 compared with the control group, #p < .05 compared with the ox-LDL group, **p < .05 compared with the aspirin group as determined by one-way ANOVA and subsequent Student–Newman–Keuls q-test (n = 5). (B. C) Protein abundance was measured by western blotting. *p < .05 compared with the control group, #p < .05 compared with the ox-LDL group, **p < .05 compared with the aspirin group, as determined by one-way ANOVA and subsequent Student–Newman–Keuls q-test (n = 5). (D, E) Abundance of IL-6 and MCP-1 in cell culture supernatant was measured by ELISAs. *p < .05 compared with the control group, #p < .05 compared with the ox-LDL group, **p < .05 compared with the aspirin group as determined by one-way ANOVA and subsequent Student–Newman–Keuls q-test (n = 5). (F) Effect of corilagin on the MOVAS cells proliferation. *p < .05 compared with the control group, #p < .05 compared with the ox-LDL group determined by one-way ANOVA and subsequent Student–Newman–Keuls q-test (n = 5). (G, H) apoptosis ratio of MOVAS were detected by Annexin V staining. No significant difference was found among four groups determined by one-way ANOVA and subsequent Student–Newman–Keuls q-test (n = 5).

Article Snippet: Afterwards, the cells were subjected to a 2-h incubation at 37°C with either primary anti-TLR4 antibody (1:200 dilution; Cat No. BA1717; Boster Biological Technology, Pleasanton, CA, USA) or anti-MyD88 antibody (1:400 dilution; Cat No. PB9148; Boster Biological Technology, Pleasanton, CA, USA).

Techniques: Expressing, Quantitative RT-PCR, Control, Quantitative Proteomics, Western Blot, Cell Culture, Staining