Journal: Brain research bulletin

Article Title: CircSATB2 modulates fear extinction memory via Robo3-driven synaptic plasticity.

doi: 10.1016/j.brainresbull.2024.111167

Figure Lengend Snippet: Fig. 5. Robo3 knockdown leads to impairment of EXT memory formation. (A) Schematic of the behavioral protocol used to test the effect of Robo3 shRNA lentivirus on EXT memory. (B) Representative image of viral infection of Robo3 shRNA lentivirus into the ILPFC. Scale bar = 1000 μm. (C) RT-qPCR shows significant Robo3 mRNA knockdown following Robo3 shRNA injection in the ILPFC (n = 8 biologically independent animals per group, two-tailed unpaired Student’s t-test, **p = 0.0088). (D) There was no effect of Robo3 knockdown on EXT learning performance. (E) Robo3 knockdown led to a significant promotes in EXT memory (n = 8 biologically independent animals per group, two-way ANOVA, F (1, 56) = 300.8, p < 0.0001; ˇSíd´ak’s multiple comparisons test: RC Control vs EXT Control, * ** *p < 0.0001; EXT Control vs EXT shRobo3, *p = 0.0109). Error bars represent SEM.

Article Snippet: The membranes were incubated overnight at 4 ◦C with either ROBO3 (Proteintech, 20220–1-AP) or GAPDH antibodies (Abclonal, AC002).

Techniques: Knockdown, shRNA, Infection, Quantitative RT-PCR, Injection, Two Tailed Test, Control

Journal: Brain research bulletin

Article Title: CircSATB2 modulates fear extinction memory via Robo3-driven synaptic plasticity.

doi: 10.1016/j.brainresbull.2024.111167

Figure Lengend Snippet: Fig. 6. Robo3 knockdown resulted in a decrease in the total number of dendritic spines. (A) Representative images of dendritic spines labelled by the Sparse Neuron Labeling Method. Scale bar = 2 μm. (B) The number of dendritic spines was significantly reduced after ILPFC injection of Robo3 shRNA (n = 20 biologically independent animals per group, two-tailed unpaired Student’s t-test, ****p < 0.0001).

Article Snippet: The membranes were incubated overnight at 4 ◦C with either ROBO3 (Proteintech, 20220–1-AP) or GAPDH antibodies (Abclonal, AC002).

Techniques: Knockdown, Labeling, Injection, shRNA, Two Tailed Test

Journal: bioRxiv

Article Title: CRISPR Screening in Tandem with Targeted mtDNA Damage Reveals WRNIP1 Essentiality

doi: 10.1101/2023.10.03.560559

Figure Lengend Snippet: (a) Comet assay detecting basal nuclear DNA damage in WRNIP1 KO cells versus WT. Quantification of comet tail DNA % is measured by ImageJ using microscopy images (n = 3 discrete replicates). Data points represent each individual cell analyzed. (b) Detection of γH2AX foci representing nuclear DNA damage in Dox and mtDox treated cells. Microscopy images comparing 24-hour treatment of HCT116 TP53 (-/-) cells with 8 µM mtDox and 25 nM Dox with and without 10 mM N -acetyl cysteine (NAC) antioxidant treatment. Moderate γH2AX detection in mtDox treated cells is attenuated by antioxidant treatment, while cells treated with Dox still display pronounced nuclear DNA damage in the presence of antioxidant. (c) Clonogenic survival comparing representative survival of cell lines treated with 8 μM mtDox. KO-1 and KO-2 represent two monoclonal WRNIP1 KO lines of HCT116 TP53 (-/-) (WT). The reintroduction of a WRNIP1 construct shows partial but incomplete recovery of the monoclonal KO lines to WT standard (n = 4, p -values are compared to WT, * p < 0.00001, ** p < 0.009) All p -values determined using unpaired t -test. Data represented as mean ± s.d.

Article Snippet: HCT116 TP53 (-/-) cells were transfected with a FLAG-tagged RIG-I expression vector purchased from Origene (RC217615) using Lipofectamine 3000 in six 150 mm dishes plated to 90% confluency the day before.

Techniques: Single Cell Gel Electrophoresis, Microscopy, Construct

Journal: Nature Cell Biology

Article Title: SEL1L–HRD1 endoplasmic reticulum-associated degradation controls STING-mediated innate immunity by limiting the size of the activable STING pool

doi: 10.1038/s41556-023-01138-4

Figure Lengend Snippet: a , qPCR analysis in primary macrophages treated with Pam3 (TLR2) or LPS (TLR4) or transfected with double-stranded RNA (RIG-I) or cGAMP (STING) ( n = 4 mice each pooled from two independent repeats). mRNA, messenger RNA. b , ELISA analysis of secreted IFNβ and TNFα in primary macrophages treated with cGAMP for 6 h ( n = 3, 3, 7 and 8 mice (left to right), pooled from three independent repeats). c , Immunoblot analysis in primary macrophages transfected with vehicle (Veh) or cGAMP for 3 h, representative of four independent repeats. d , Immunoblot analysis in primary macrophages treated with vehicle or DMXAA for 1.5 h, representative of four independent repeats. e , ELISA analysis of secreted IFNβ and TNFα in primary macrophages transfected with poly(I:C) (RIG-1) for 18 h ( n = 3, 3, 4 and 4 mice (left to right); combined from two independent repeats). f , Immunoblot analysis in primary macrophages transfected with vehicle or poly(I:C) for 6 h, representative of two independent repeats. The relative intensity of p-STING (normalized to β-tubulin) or ratio of phosphorylated to total protein (p/t) are shown below the blots ( c , d and f ). All values represent means ± s.e.m. Statistical significance was determined by unpaired, two-tailed Student’s t -test ( a , b and e ).

Article Snippet: Inflammatory stimuli included the TLR2 ligand Pam3Cy (InvivoGen) at 1 μg ml −1 , the TLR4 ligand LPS (InvivoGen) at 500 ng ml −1 , the STING ligands 2′3′-cGAMP (InvivoGen), c-di-AMP (InvivoGen) and DMXAA (MedChemExpress) at 3.5 μg ml −1 , 3.5 μg ml −1 and 20 μg ml −1 , respectively, and the RIG-1 ligand poly(I:C) (InvivoGen) at 2 μg ml −1 .

Techniques: Transfection, Enzyme-linked Immunosorbent Assay, Western Blot, Two Tailed Test

Journal: American Journal of Cancer Research

Article Title: RARRES3 suppressed metastasis through suppression of MTDH to regulate epithelial-mesenchymal transition in colorectal cancer

doi:

Figure Lengend Snippet: RARRES3 was significantly down-regulated in CRC tissues. Shown were immunohistochemical analysis of the expression of RARRES3 in CRC tissues. A. Strong expression of RARRES3. B. Moderate expression of RARRES3. C. Weak expression of RARRES3. D. Negative expression of RARRES3 in CRC tissues. E and F. Were immunohistochemical analysis of the expression of RARRES3 in paired normal tissues (× 200).

Article Snippet: Additionally, eukaryotic over-expression vector harboring full length cDNA of RARRES3 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_004585.2","term_id":"8051633","term_text":"NM_004585.2"}} NM_004585.2 ) pCMV-XL5-RARRES3 (SC 117294) was also purchased from Origene (Origene Technologies, Inc, USA).

Techniques: Immunohistochemical staining, Expressing

Journal: American Journal of Cancer Research

Article Title: RARRES3 suppressed metastasis through suppression of MTDH to regulate epithelial-mesenchymal transition in colorectal cancer

doi:

Figure Lengend Snippet: Correlation between RARRES3 expression and clinicopathologic information of CRC

Article Snippet: Additionally, eukaryotic over-expression vector harboring full length cDNA of RARRES3 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_004585.2","term_id":"8051633","term_text":"NM_004585.2"}} NM_004585.2 ) pCMV-XL5-RARRES3 (SC 117294) was also purchased from Origene (Origene Technologies, Inc, USA).

Techniques: Expressing

Journal: American Journal of Cancer Research

Article Title: RARRES3 suppressed metastasis through suppression of MTDH to regulate epithelial-mesenchymal transition in colorectal cancer

doi:

Figure Lengend Snippet: RARRES3 expression wasn’t significantly associated with prognosis in CRC. Immunostaining of RARRES3 and overall survival in CRC was calculated by the Kaplan-Meier survival curves. There wasn’t significant difference between RARRES3 expression and prognosis (P>0.05, using log rank test) after statistical analysis.

Article Snippet: Additionally, eukaryotic over-expression vector harboring full length cDNA of RARRES3 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_004585.2","term_id":"8051633","term_text":"NM_004585.2"}} NM_004585.2 ) pCMV-XL5-RARRES3 (SC 117294) was also purchased from Origene (Origene Technologies, Inc, USA).

Techniques: Expressing, Immunostaining

Journal: American Journal of Cancer Research

Article Title: RARRES3 suppressed metastasis through suppression of MTDH to regulate epithelial-mesenchymal transition in colorectal cancer

doi:

Figure Lengend Snippet: RARRES3 could suppress EMT process while involving the up-regulation of β-catenin. A. Knock-down efficiency of candidate shRNA target sequences was determed by western-blotting. Both the 3 different shRNA vectors as well as shRNA control (scramble) vetor were transfected in CRC cell line HT-29. shRNA-RARRES3-2 was chosen as the best which was abbreviated for shRNA-RARRES3 in the following funcational exmperiment. B. The endogenous expression of RARRES3 in a panel of CRC cell lines was detected using immunoblotting. β-Tubulin was loading control. 80μg total protein was loaded per lane, which was separated by 10% SDS-PAGE followed by visualization with Western Breeze kit (Invitrogen, USA). C. Immunoblotting assay for HT-29 and SW-620 after knock-down and over-expression of RARRES3 respectively using retroviral shRNA and over-expression vector transfection for 96 hours.

Article Snippet: Additionally, eukaryotic over-expression vector harboring full length cDNA of RARRES3 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_004585.2","term_id":"8051633","term_text":"NM_004585.2"}} NM_004585.2 ) pCMV-XL5-RARRES3 (SC 117294) was also purchased from Origene (Origene Technologies, Inc, USA).

Techniques: Knockdown, shRNA, Western Blot, Control, Transfection, Expressing, SDS Page, Over Expression, Retroviral, Plasmid Preparation

Journal: American Journal of Cancer Research

Article Title: RARRES3 suppressed metastasis through suppression of MTDH to regulate epithelial-mesenchymal transition in colorectal cancer

doi:

Figure Lengend Snippet: RARRES3 could suppress both the invasion and migration ability but not proliferation in CRC cell lines. A. MTT assay for HT-29 and SW-620 after knock-down of RARRES3 using over-expression vector and retroviral shRNA vector transfection for 0 h, 24 h, 48 h, 72 h and 96 h; B. Transwell assays for HT-29 and SW-620 after knock-down and over-expression of RARRES3 using retroviral shRNA vector and over-expression vector transfection for 48 hours. The left is qualification assay; the right is quantitation assay of Transwell; C. Wound-healing assay for HT-29 and SW-620 after knock-down and over-expression of RARRES3 using retroviral shRNA vector and over-expression vector transfection for 96 hours. (**P<0.01, *P<0.05 compared with control group using independent Student’s t test or one-way ANOVA analysis). Images of migratory/Invasion cells from the scratched boundary/transwell chamber were observed and acquired with light microscope (10 × 10). Similar results were obtained in three independent experiments, and shown were representative figures.

Article Snippet: Additionally, eukaryotic over-expression vector harboring full length cDNA of RARRES3 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_004585.2","term_id":"8051633","term_text":"NM_004585.2"}} NM_004585.2 ) pCMV-XL5-RARRES3 (SC 117294) was also purchased from Origene (Origene Technologies, Inc, USA).

Techniques: Migration, MTT Assay, Knockdown, Over Expression, Plasmid Preparation, Retroviral, shRNA, Transfection, Quantitation Assay, Wound Healing Assay, Control, Light Microscopy

Journal: American Journal of Cancer Research

Article Title: RARRES3 suppressed metastasis through suppression of MTDH to regulate epithelial-mesenchymal transition in colorectal cancer

doi:

Figure Lengend Snippet: RARRES3 interacted with MTDH in an opposite way. A. Expression variation of MTDH, β-catenin, Fibronectin, E-cadherin after ectopic expression of RARRES3 in HT-29 and SW-620 where basal expression of RARRES3 was highest and lowest respectively; B. Expression of β-catenin both in cytoplasmic and nuclear compartment was detected using western-blotting; GAPDH and Histone H4 were as loading control. 100 μg total protein was loaded per lane, which was separated by 10% SDS-PAGE followed by visualization with Western Breeze kit (Invitrogen, USA). C. Immunoprecipitation analysis of RARRES3 and MTDH. 293T cells transfected with the eukaryotic expression vector of RARRES3 that contains Flag tag on N-terminus were harvested for immunoprecipitation (IP) using anti-FLAG M2-agarose beads. FLAG precipitates were subjected for immunoblot using anti-RARRES3, anti-MTDH, or anti-GAPDH antibody.

Article Snippet: Additionally, eukaryotic over-expression vector harboring full length cDNA of RARRES3 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_004585.2","term_id":"8051633","term_text":"NM_004585.2"}} NM_004585.2 ) pCMV-XL5-RARRES3 (SC 117294) was also purchased from Origene (Origene Technologies, Inc, USA).

Techniques: Expressing, Western Blot, Control, SDS Page, Immunoprecipitation, Transfection, Plasmid Preparation, FLAG-tag

Journal: American Journal of Cancer Research

Article Title: RARRES3 suppressed metastasis through suppression of MTDH to regulate epithelial-mesenchymal transition in colorectal cancer

doi:

Figure Lengend Snippet: RARRES3 could suppress colorectal cancer (CRC) metastasis in vivo. RARRES3 could suppress colorectal cancer (CRC) metastasis in vivo. Representative livers derived from severe combined immunodeficient (BALB/c) mice in HT-29 (A) and SW-620 (B) were shown. The formation of metastatic nodules at the liver surface could be significantly promoted by HT-29-shRNA-RARRES3 and significantly suppressed by SW-620-pCMV-RARRES3.

Article Snippet: Additionally, eukaryotic over-expression vector harboring full length cDNA of RARRES3 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_004585.2","term_id":"8051633","term_text":"NM_004585.2"}} NM_004585.2 ) pCMV-XL5-RARRES3 (SC 117294) was also purchased from Origene (Origene Technologies, Inc, USA).

Techniques: In Vivo, Derivative Assay, shRNA

Journal: Acta neuropathologica

Article Title: SMN regulates GEMIN5 expression and acts as a modifier of GEMIN5-mediated neurodegeneration

doi: 10.1007/s00401-023-02607-8

Figure Lengend Snippet: Smn upregulation suppresses loss of Gemin5 neurodegeneration in vivo. a Schematic representing the experimental to investigating the genetic manipulation of the Drosophila snRNP complex proteins in the background of loss of Gemin5 (Rig) animals in vivo. Drosophila expressing UAS-Rig RNAi were crossed with the GMR-gal4 driver for targeted expression to the Drosophila eye and Tubulin-gal4 driver for ubiquitous expression. b Representative images of female Drosophila eyes expressing GMR-gal4 and Rig RNAi crossed with control (W1118), Luciferase OE, Smn RNAi, and Smn OE. c, d, e Quantification of eye degeneration of control and Rig RNAi flies combined with (c) Luciferase OE, (d) Smn RNAi, and (e) Smn OE (n = 20 Drosophila per group, one-way ANOVA w/ Tukey’s multiple comparisons). Error bars indicate S.E.M. ****p < 0.0001. NS, not significant. f Quantification of relative eye sizes of control and Rig RNAi flies combined with Smn OE (n = 5 Drosophila per group, one-way ANOVA w/ Tukey’s multiple comparisons). Error bars indicate S.E.M. *p < 0.05; ****p < 0.001. NS, not significant. g, h qPCR analysis from (n = 4 biological replicates of 6 Drosophila heads per group) confirms significant knockdown of endogenous (g) Rig in the Rig KD groups and significant overexpression of (h) Smn in the Smn OE crosses (one-way ANOVA w/ Tukey’s multiple comparisons). Error bars indicate S.E.M. *p < 0.05; ****p < 0.0001; NS, not significant. i Representative male images of eclosed Drosophila adults expressing Control, Rig RNAi (pupal lethality), Rig RNAi in combination with over expression of Smn, and Smn OE. j The percentage of eclosed adults for control and Rig RNAi animals combined with Smn OE (n = 100 Drosophila per replicate, one-way ANOVA w/ Tukey’s multiple comparisons). Error bars indicate S.E.M. ****p < 0.0001

Article Snippet: Western blots were blocked with 2.5% milk solution (BLOT-QuickBlocker reagent, EMB Millipore, #WB57–175GM) and incubated with primary antibody overnight: rabbit Drosophila Rig, 1–1:000 (developed by Bio Boster Biological Technology Co. Ltd, Wuhan, China), rabbit Drosophila Smn (developed by Bio Boster Biological Technology Co. Ltd, Wuhan, China), and mouse anti-tubulin, 1:10,000 (66,031, Protein Tech).

Techniques: In Vivo, Expressing, Control, Luciferase, Knockdown, Over Expression

Journal: Acta neuropathologica

Article Title: SMN regulates GEMIN5 expression and acts as a modifier of GEMIN5-mediated neurodegeneration

doi: 10.1007/s00401-023-02607-8

Figure Lengend Snippet: SMN expression alleviates reduced snRNP biogenesis in GEMIN5-mediated disease. a Confocal images of control and mutant GEMIN5 L1068P neurons transduced with EGFP and lentiviral EGFP-SMN from the same set of neuronal differentiations. The neurons were probed for the snRNP marker Y12 (Sm), GFP, and the neuronal marker microtubule-associated protein 2 (MAP2). The cell nuclei were stained with DAPI. Scale bar = 10 μm. b Quantitative analysis observing a significant decrease in nuclear snRNP expression in L1068P neurons after lentiviral transduction of EGFP. Lentiviral transduction of EGFP-SMN significantly rescues the levels of nuclear snRNP expression in L1068P neurons (n = 75–100 neurons, one-way ANOVA w/ Tukey’s multiple comparisons). Error bars indicate S.E.M. *p < 0.05; **p < 0.01; ****p < 0.0001. NS, not significant. c Confocal images of control and mutant GEMIN5 H913R neurons transduced with EGFP and lentiviral EGFP-SMN from the same set of neuronal differentiations. The neurons were probed for the small nuclear ribonucleoprotein interacting partner, Coilin (cajal bodies, CBs), GFP, and GEMIN5. The cell nuclei were stained with DAPI. Scale bar = 10 μm. d Quantitative analysis observing the average number of CBs per cell in control and mutant H913R neuronal cells with EGFP and EGFP-SMN lentiviral expression. Only CBs above 0.2 microns were quantified (n = 60–80 neurons, one-way ANOVA w/ Tukey’s multiple comparisons). Error bars indicate S.E.M. **p < 0.01; NS, not significant. e The percentage of cells with CBs in control and mutant H913R neuronal cells with EGFP and EGFP-SMN expression (n = 60–80 neurons, one-way ANOVA w/ Bonferroni test). Error bars indicate S.E.M. **p < 0.01; NS, not significant. f Representative gel displaying complete in vitro snRNP assembly formation using 3′ Cy3-biotin-labeled U1 snRNA and the cytoplasmic extract from control and L1068P neurons with lentirviral SMN and ASO N1 administration. Untransfected HEK293T cells and HEK293T transfected with GEMIN5 shRNA were used as a positive control for assembly formation. g Representative quantification of 3 biological replicate sample displaying the intensity of the snRNP assembly between groups (n = 3, one-way ANOVA w/ Tukey’s multiple comparisons). Error bars indicate S.E.M. *p < 0.05; **p < 0.01. NS, not significant. h Confocal images of the ventral nerve cord (VNC) of control and ubiquitously expressing GEMIN5 KD Drosophila larvae with and without Smn over expression stained with the snRNP marker Sm (Y12) and nuclei (DAPI). Loss of GEMIN5 in vivo results in a reduction of nuclear Sm which is rescued by upregulation of Smn. Scale bar = 10 μM. i. Confocal images of the ventral nerve cord (VNC) of control and ubiquitously expressing GEMIN5 KD Drosophila larvae with and without Smn over expression stained with the small nuclear ribonucleoprotein interacting partner, dCoilin (CBs) and nuclei (DAPI). Loss of GEMIN5 in vivo results in a drastic reduction of CB formation which is rescued by upregulation of Smn. Scale bar = 10 μM

Article Snippet: Western blots were blocked with 2.5% milk solution (BLOT-QuickBlocker reagent, EMB Millipore, #WB57–175GM) and incubated with primary antibody overnight: rabbit Drosophila Rig, 1–1:000 (developed by Bio Boster Biological Technology Co. Ltd, Wuhan, China), rabbit Drosophila Smn (developed by Bio Boster Biological Technology Co. Ltd, Wuhan, China), and mouse anti-tubulin, 1:10,000 (66,031, Protein Tech).

Techniques: Expressing, Control, Mutagenesis, Transduction, Marker, Staining, In Vitro, Labeling, Transfection, shRNA, Positive Control, Over Expression, In Vivo

Journal: International Journal of Molecular Sciences

Article Title: Activation of Endothelial Large Conductance Potassium Channels Protects against TNF-α-Induced Inflammation

doi: 10.3390/ijms24044087

Figure Lengend Snippet: Protein-protein interaction (PPI) prediction model (STRING): ( A ) By entering KCNMA1 (BK channel), CACNA1 (Ca V channel), TNF-α, and CCL2 as query proteins (in red boxes), the STRING PPI network model reveals the nine most likely downstream interaction partners with the confidence level set to “high” (0.8 on a 0–1 scale). ( B ) STRING model of cluster analysis using K-MEANS algorithm to categorize each protein molecule into a determined sub-cluster: Ion channel, Cytokine, TNF-α pathway. ( C , D ) KEGG pathway analysis in STRING, highlighting relevant pathways in red (RIG-1), blue (NF-kB-1), and green (MAPK). The “strength” reflects the score of integrated, publicly available PPI information: the higher the “strength” number, the more likely this pathway is present in the model. “False discovery rate” (FDR) is based on a statistical approach used in high-throughput experiments to correct for random events that falsely appear significant: the lower the FDR, the more likely a pathway is involved in a given network. All proteins in this figure are labeled with their gene names, as is conventional in this field. The red dashed line depicts the association between Ca V channels and CCL-2 identified in this study.

Article Snippet: To validate the activation of our top 3 predicted signaling pathways (RIG-1, NF-kB, MAPK), we used an ELISA-based RIG-1-like receptor (RLR) kit (MyBioSource, San Diego, CA; #MBS262313), an NF-kB/p65 (pS536 + Total) detection kit (Abcam; #ab176663), a multiplex immunoassay MAP kinase phosphoprotein (phospho-p38, phospho-ERK-1/2, phospho-JNK-1/2) kit, and an electrochemiluminescent platform (Meso Scale Discovery, MSD, Rockville, Maryland #K15101D-1), respectively [ , ].

Techniques: High Throughput Screening Assay, Labeling

Journal: International Journal of Molecular Sciences

Article Title: Activation of Endothelial Large Conductance Potassium Channels Protects against TNF-α-Induced Inflammation

doi: 10.3390/ijms24044087

Figure Lengend Snippet: BK channel activation inhibits TNF-α-induced MAPK but not NF-kB-1/p65 or RIG-1 pathways. The treatment of HULEC5a endothelial cells with the BK channel opener NS1619 (30 µM, for 24 h) inhibits the TNF-α-induced (5 ng/mL; 24 h) phosphorylation of the Ca 2+ -dependent JNK-1/2 ( A ) and p38 pathways ( B ), but has no effect on RIG-1 expression or the phosphorylation of ERK-1/2 and NF-kB/p65 ( C – F ) ( n = 3–4, * p ≤ 0.05) as shown by ELISA-based multiplex assays. ( G ) The inhibition of JNK-1/2 (JNK inhibitor VIII, 20 µM, 24 h) and p38 (SB203580, 20 µM, 24 h) pathways, with or without NS1619, decreases TNF-α-induced (5 ng/mL; 24 h) CCL-2 secretion from HULEC5a cells ( n = 3–4, * p ≤ 0.0001), shown by ELISA. n = number of separate cell passages, which are considered biological replicates.

Article Snippet: To validate the activation of our top 3 predicted signaling pathways (RIG-1, NF-kB, MAPK), we used an ELISA-based RIG-1-like receptor (RLR) kit (MyBioSource, San Diego, CA; #MBS262313), an NF-kB/p65 (pS536 + Total) detection kit (Abcam; #ab176663), a multiplex immunoassay MAP kinase phosphoprotein (phospho-p38, phospho-ERK-1/2, phospho-JNK-1/2) kit, and an electrochemiluminescent platform (Meso Scale Discovery, MSD, Rockville, Maryland #K15101D-1), respectively [ , ].

Techniques: Activation Assay, Expressing, Enzyme-linked Immunosorbent Assay, Multiplex Assay, Inhibition