Journal: Brain, behavior, and immunity

Article Title: Distinct cellular mediators drive the Janus Faces of Toll-like Receptor 4 regulation of network excitability which impacts working memory performance after brain Injury

doi: 10.1016/j.bbi.2020.03.035

Figure Lengend Snippet: Antibodies

Article Snippet: Densitometric quantification was determined using Image-J software (NIH) and normalized to β-actin density. table ft1 table-wrap mode="anchored" t5 Table 1: caption a7 Antibody Molecular Type Weight Dilution Company Catalog Number Figure # MAP2 Chicken Polyclonal 1:1000 AbCam ab5392 GFAP Mouse monoclonal 1:1000 Millipore MAB360 Data not shown IBA-1 Mouse monoclonal 1:1000 Millipore MABN92 Data not shown β-actin 42kDa Mouse Monoclonal 1:5000 Sigma-Aldrich A1978 HMGB1 29kDa Rabbit Polyclonal 1:1000 Abcam AB18256 MyD88 35kDa Rabbit Polyclonal 1:1000 Abcam Ab2064 IKB 40kDa Mouse Monoclonal 1:500 Novus 6A920 (NB100-56507) NFkB 60kDa Rabbit Polyclonal 1:1000 Abcam Ab16502 TICAM2 32kDa Rabbit Polyclonal 1:500 Millipore ABF235 IRF3 47kDa Rabbit Polyclonal 1:1000 Abcam Ab25950 TNFa 25kDa Rabbit Polyclonal 1:1000 Abcam Ab9635 Open in a separate window Antibodies 2.6.

Techniques:

Journal: Brain, behavior, and immunity

Article Title: Distinct cellular mediators drive the Janus Faces of Toll-like Receptor 4 regulation of network excitability which impacts working memory performance after brain Injury

doi: 10.1016/j.bbi.2020.03.035

Figure Lengend Snippet: Representative western blots of HMGB1 (A) MyD88, IkBα, and NFκB, (C) and, TICAM2, IRF3 and TNFα (G) in hippocampal samples from the injured side obtained 3 days after vehicle/CLI-095 treatment. Treatments were started 24 hours after injury. Corresponding β-actin bands are illustrated. (A, C and G) Summary histograms of expression of HMGB1 (B), MyD88 (D), IkBα (E), NFκB (F), TICAM2 (H), IRF3 (I) and TNFα (J), normalized to the expression levels in sham-vehicle treated controls. * indicates p<0.05 compared to sham and # indicates p<0.05 compared to corresponding ACSF by TW ANOVA followed by post-hoc Tukey’s test.

Article Snippet: Densitometric quantification was determined using Image-J software (NIH) and normalized to β-actin density. table ft1 table-wrap mode="anchored" t5 Table 1: caption a7 Antibody Molecular Type Weight Dilution Company Catalog Number Figure # MAP2 Chicken Polyclonal 1:1000 AbCam ab5392 GFAP Mouse monoclonal 1:1000 Millipore MAB360 Data not shown IBA-1 Mouse monoclonal 1:1000 Millipore MABN92 Data not shown β-actin 42kDa Mouse Monoclonal 1:5000 Sigma-Aldrich A1978 HMGB1 29kDa Rabbit Polyclonal 1:1000 Abcam AB18256 MyD88 35kDa Rabbit Polyclonal 1:1000 Abcam Ab2064 IKB 40kDa Mouse Monoclonal 1:500 Novus 6A920 (NB100-56507) NFkB 60kDa Rabbit Polyclonal 1:1000 Abcam Ab16502 TICAM2 32kDa Rabbit Polyclonal 1:500 Millipore ABF235 IRF3 47kDa Rabbit Polyclonal 1:1000 Abcam Ab25950 TNFa 25kDa Rabbit Polyclonal 1:1000 Abcam Ab9635 Open in a separate window Antibodies 2.6.

Techniques: Western Blot, Expressing

Journal: Scientific Reports

Article Title: Innate immune responses through Toll-like receptor 3 require human-antigen-R-mediated Atp6v0d2 mRNA stabilization

doi: 10.1038/s41598-019-56914-w

Figure Lengend Snippet: Defective response to TLR3 in HuR KO cells ( a ) Cell lysates from wild-type (WT), HuR KO1, and HuR KO2 cells were subjected to western blotting (WB) and probed with anti-HuR and anti-actin antibodies. ( b ) WT, HuR KO1, and HuR KO2 cells were stimulated with poly(I:C), R837, or ODN1668 for 8 h, and Ifnb1 and Cxcl10 mRNA expression were measured with RT–qPCR. ( c ) WT and HuR KO1 cells were stimulated for the indicated times, and the cell lysates were subjected to WB and probed with an anti-pIRF3, anti-IRF3, anti-pI k Bα or anti-I k Bα antibody. ( d ) HuR KO1 cells were stably transfected with FLAG–HuR-expressing plasmid with retroviral infection. Lysates from WT, HuR KO1, and HuR KO1 + FLAG–HuR cells were subjected to WB and probed with anti-FLAG, anti-HuR, or anti-actin antibody. ( e ) These cells were stimulated with poly(I:C) and the expression levels of Ifnb1 and Cxcl10 mRNAs were quantified with RT–qPCR. Data are the means ± SE of triplicate independent experiments. *p < 0.01, Student’s t test.

Article Snippet: Mouse anti-HuR monoclonal antibody (mAb; 3A2; Santa Cruz Biotechnology), rabbit anti-IRF3 monoclonal antibody (D83B9; Cell Signaling Technology), rabbit anti-phospho-IRF3 (Ser396) monoclonal antibody (4DaG; Cell Signaling Technology), rabbit anti-NF-κB p65 monoclonal antibody (D14E12; Cell Signaling Technology), rabbit anti-IRF3 polyclonal antibody (FL-425; Santa Cruz Biotechnology), rabbit anti-phospho-IκBα (Ser32) monoclonal antibody (14D4; Cell Signaling Technology), mouse anti-IκBα monoclonal antibody (L35A5; Cell Signaling Technology), goat anti-actin polyclonal antibody (I-19; Santa Cruz Biotechnology), and mouse anti-FLAG M2 monoclonal antibody (Sigma-Aldrich) were purchased as commercially available products.

Techniques: Western Blot, Expressing, Quantitative RT-PCR, Stable Transfection, Transfection, Plasmid Preparation, Retroviral, Infection

Journal: Scientific Reports

Article Title: Innate immune responses through Toll-like receptor 3 require human-antigen-R-mediated Atp6v0d2 mRNA stabilization

doi: 10.1038/s41598-019-56914-w

Figure Lengend Snippet: HuR regulates Atp6v0d2 mRNA expression and endosomal acidification. ( a ) Expression for Tlr3 , Traf3 and Irf3 in WT and HuR KO1 were measured by RT-qPCR ( b , c ) HuR KO1 and KO2 cells were stimulated with poly(I:C), R837 or ODN1668 and the levels of Atp6v0d2 ( b ), Atp6v1a , and Atp6v1b2 ( c ) were quantified with RT–qPCR. ( d ) HuR KO1 cells stably expressed FLAG–HuR after retroviral infection. Wild-type ( WT), HuR KO1, and HuR KO1 + FLAG–HuR cells were stimulated with poly(I:C) and Atp6v0d2 mRNA expression was measured with RT–qPCR. ( e ) Endosomal acidification was visualized with acridine orange staining. Red dots indicate acidified endosomes, highlighted with white arrowhead. Top panel: WT cells; middle panel: WT cells treated with bafilomycin A1; lower panel: HuR KO1 cells. Scale bar, 10 µm. ( f ) Number of red positive cells was counted and the percentage of positive cells was plotted as bar graph. Data are the means ± SE of triplicate independent experiments. *p < 0.01, Student’s t test.

Article Snippet: Mouse anti-HuR monoclonal antibody (mAb; 3A2; Santa Cruz Biotechnology), rabbit anti-IRF3 monoclonal antibody (D83B9; Cell Signaling Technology), rabbit anti-phospho-IRF3 (Ser396) monoclonal antibody (4DaG; Cell Signaling Technology), rabbit anti-NF-κB p65 monoclonal antibody (D14E12; Cell Signaling Technology), rabbit anti-IRF3 polyclonal antibody (FL-425; Santa Cruz Biotechnology), rabbit anti-phospho-IκBα (Ser32) monoclonal antibody (14D4; Cell Signaling Technology), mouse anti-IκBα monoclonal antibody (L35A5; Cell Signaling Technology), goat anti-actin polyclonal antibody (I-19; Santa Cruz Biotechnology), and mouse anti-FLAG M2 monoclonal antibody (Sigma-Aldrich) were purchased as commercially available products.

Techniques: Expressing, Quantitative RT-PCR, Stable Transfection, Retroviral, Infection, Staining

Journal: Scientific Reports

Article Title: Innate immune responses through Toll-like receptor 3 require human-antigen-R-mediated Atp6v0d2 mRNA stabilization

doi: 10.1038/s41598-019-56914-w

Figure Lengend Snippet: Reduced TLR3 response in ATP6V0D2 KO cells. ATP6V0D2 KO cells exogenously expressed FLAG–ATP6V0D2 after retroviral infection. After puromycin selection, Atp6v0d2 expression was detected with western blotting (WB) ( a ) and RT–qPCR ( b ). ( c ) Following stimulation with poly(I:C) for 8 h, Ifnb1 and Cxcl10 expression was quantified with RT–qPCR. ( d ) WT and ATP6V0D2 KO cells were stimulated for the indicated times, and the cell lysates were subjected to WB with an anti-pIRF3, anti-IRF3, anti-pI k Bα or anti-I k Bα antibody. Data are the means ± SE of triplicate independent experiments. *p < 0.01, Student’s t test.

Article Snippet: Mouse anti-HuR monoclonal antibody (mAb; 3A2; Santa Cruz Biotechnology), rabbit anti-IRF3 monoclonal antibody (D83B9; Cell Signaling Technology), rabbit anti-phospho-IRF3 (Ser396) monoclonal antibody (4DaG; Cell Signaling Technology), rabbit anti-NF-κB p65 monoclonal antibody (D14E12; Cell Signaling Technology), rabbit anti-IRF3 polyclonal antibody (FL-425; Santa Cruz Biotechnology), rabbit anti-phospho-IκBα (Ser32) monoclonal antibody (14D4; Cell Signaling Technology), mouse anti-IκBα monoclonal antibody (L35A5; Cell Signaling Technology), goat anti-actin polyclonal antibody (I-19; Santa Cruz Biotechnology), and mouse anti-FLAG M2 monoclonal antibody (Sigma-Aldrich) were purchased as commercially available products.

Techniques: Retroviral, Infection, Selection, Expressing, Western Blot, Quantitative RT-PCR

Journal: Virology

Article Title: MicroRNA-23 inhibits PRRSV replication by directly targeting PRRSV RNA and possibly by upregulating type I interferons.

doi: 10.1016/j.virol.2013.12.020

Figure Lengend Snippet: Fig. 6. miR-23 increases type I interferon expression through IRF3/IRF7 activation during PRRSV infection. (A, B and C) IRF3/IRF7 inhibitor impaired the induction of type I IFNs during PRRSV infection. Transfection of miR-23 mimics or NC in PAMs was performed prior to the treatment of the indicated inhibitors or DMSO, followed by PRRSV JXwn06 infection for 48 h (MOI¼0.01). Cells were then harvested for quantifying the expression of type I interferon genes IFN-β (A) and IFN-α (B), and ORF7 (C) using qRT- PCR, normalized to GAPDH. The data were represented as fold changes of the indicated genes after over-expression of miR-23 (NC was set up as 1 and not shown in the figure). Statistical significance was analyzed by one-way ANOVA followed by post hoc Dunnett0s multiple comparison. Significance compared to DMSO-baseline: nPo0.05. (D, E, F, and G) miR-23 increases activation of IRF3 during PRRSV infection. Either miR-23 or NC mimics and pRL-TK were co-transfected with IRF3 (D), ISRE (E), or NF-κB (F) luciferase reporters into Marc-145 cells. Cells were then either infected with CH-1a (MOI¼0.01) 6 h after transfection, or transfected with 1 μg poly(I:C) 24 h post trasfection, or left untreated (mock). All cells were harvested 36 h post-transfection for dual-luciferase assay. (G) Western blot analysis of phospho-IRF3 or IRF3 in PAMs transfected with miR-23 mimics followed by infection with JXwn06 (MOI¼0.01) for 48 h. β-actin was shown as a loading control. (H) miR-23 plays no role in IFN down- stream pathway. Luciferase activity in lysates of CRL-2843 cells co-transfected with ISRE luciferase reporter, pRL-TK and miRNA mimics for 6 h and incubated with or without 10 U/ml IFN-α for 24 h. The data were represented as fold increase of ISRE. Data were representative of three independent experiments (mean7SD). Statistical significance was analyzed by t-test; nPo0.05; ns, not significant.

Article Snippet: Membranes were blocked with 5% milk in PBS with Tween-20 (PBST 0.025% Tween-20) and probed with Rabbit anti-GP5 polyclonal antibodies (1:5000, prepared in our lab), anti-Phospho-IRF3 polyclonal antibodies (1:1000; #4947, Cell Signaling), or anti-IRF3 polyclonal antibodies (1:1000; #119041, Cell Signaling) for 1 h at room temperature.

Techniques: Expressing, Activation Assay, Infection, Transfection, Quantitative RT-PCR, Over Expression, Comparison, Luciferase, Western Blot, Control, Activity Assay, Incubation