Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: A paradox of transcriptional and functional innate interferon responses of human intestinal enteroids to enteric virus infection

doi: 10.1073/pnas.1615422114

Figure Lengend Snippet: Analysis of IFN protein from HIEs

Article Snippet: The type III IFN receptor was blocked with a polyclonal sheep antibody (25 μg/mL) against the IL28RA subunit (cat. no. AF5260, lot CCXR01; R&D Systems).

Techniques:

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: A paradox of transcriptional and functional innate interferon responses of human intestinal enteroids to enteric virus infection

doi: 10.1073/pnas.1615422114

Figure Lengend Snippet: IFN response to inactivated HRV. HIEs (j3) were inoculated with replication-competent HRV (MOI of 10 FFU) or with an equivalent amount (in micrograms) of inactivated HRV particles. (A) Expression of type III IFN (IFNL1), type I IFN (IFNB1), and the ISG IFI44L was assessed at 6 hpi by RT-qPCR. Each data bar represents the geometric mean ± 95% CI of the geometric mean (n = 4 technical replicates). Statistical analyses were performed by Mann–Whitney U test. (B) HIEs were infected as described in A and, after 1 h, HIEs were washed and resuspended in medium containing type III IFN receptor-blocking antibody (25 μg/mL anti-IL28RA). The media was analyzed at 20 hpi for secreted IFN-λ1 by ELISA. Data from one representative experiment of two independent experiments is presented as arithmetic mean ± SD of three or four technical replicates.

Article Snippet: The type III IFN receptor was blocked with a polyclonal sheep antibody (25 μg/mL) against the IL28RA subunit (cat. no. AF5260, lot CCXR01; R&D Systems).

Techniques: Expressing, Quantitative RT-PCR, MANN-WHITNEY, Infection, Blocking Assay, Enzyme-linked Immunosorbent Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: A paradox of transcriptional and functional innate interferon responses of human intestinal enteroids to enteric virus infection

doi: 10.1073/pnas.1615422114

Figure Lengend Snippet: Effect of receptor-blocking antibodies on rotavirus infectivity and induction of OAS2 transcripts. (A) Stock aliquots of Ito-HRV were incubated with no antibody (control), the anti-type III IFN receptor-blocking antibody, or the anti-type I IFN system antibody mixture for 2 h. To assess the titer of HRV under these conditions, MA104 cells were infected with control Ito-HRV aliquots or antibody-treated Ito-HRV aliquots for 1 h, washed twice, and incubated overnight in DMEM for 18 h. Viral titer was subsequently obtained by FFA. (B) HIEs (j11) suspended in differentiation medium containing 0.5 mg/mL pancreatin were treated with or without blocking antibodies to the type III IFN receptor (anti-IL28RA) or the type I IFN system (anti-type 1 IFN mixture). After 1 h, 100 U/mL of IFN-λ1 or IFN-β1 was added to the HIEs. After 26 h, total RNA was extracted, and the transcriptional response was compared with HIE cultures that were neither antibody-treated nor IFN-treated. Transcript levels of the ISG OAS2 were first normalized to GAPDH levels, and the fold increase was calculated by using the 2−ΔΔCt method. Displayed above the bars is the percent reduction in OAS2 levels between the two groups being compared. Data in A and B are presented as geometric mean ± 95% CI of the geometric mean of three technical replicates. (C) HIEs (j3) were treated with or without blocking antibodies to the type III IFN receptor (anti-IL28RA) or the type I IFN system (anti-type 1 IFN mixture). After 2 h, HIEs were treated with 100 U/mL of IFN-λ1 or IFN-β1 for 24 h. After 24 h, HIEs were washed to remove the antibodies and IFN and inoculated with Ito-HRV (MOI of 0.1). The control samples consisted of HIEs which did not receive antibodies nor IFN before Ito-HRV infection. The increase in infectious virus was calculated by subtracting the titer at 1.5 hpi in the control infection from the titer at 24 hpi in each of the five test groups. Data from one representative experiment of two independent experiments is presented as arithmetic mean ± SEM of four technical replicates. Statistical analyses were performed by Mann–Whitney U test.

Article Snippet: The type III IFN receptor was blocked with a polyclonal sheep antibody (25 μg/mL) against the IL28RA subunit (cat. no. AF5260, lot CCXR01; R&D Systems).

Techniques: Blocking Assay, Infection, Incubation, Control, Virus, MANN-WHITNEY

Journal: bioRxiv

Article Title: Identification of markers for the isolation of neuron-specific extracellular vesicles

doi: 10.1101/2024.04.03.587267

Figure Lengend Snippet: Validation of NRXN3 as neuron-specific marker present on EVs and immuno-isolation of NRXN3 + EVs. A. Simoa measurement in SEC fractions of pooled CSF of NRXN3 (top), CD9 (middle) and albumin (bottom). B. Simoa measurement in SEC fractions of pooled plasma of NRXN3 (top), CD9 (middle) and albumin (bottom). C. Simoa measurement in DGC fractions of pooled plasma of NRXN3 (top), CD9 (middle) and albumin (bottom). D. Simoa measurement of NRXN3 in SEC fractions of plasma from three different individuals. For A-D, Simoa measurements were taken in duplicate for each sample. Error bars indicate SD.

Article Snippet: The following antibodies were used for immuno-isolation of NRXN3 and controls: sheep polyclonal NRXN3 (cat. AF5269, R&D Systems), GFP (4745-1051, Rockland), and Isotype Control (AB37385, Abcam).

Techniques: Biomarker Discovery, Marker, Isolation, Clinical Proteomics

Journal: bioRxiv

Article Title: Identification of markers for the isolation of neuron-specific extracellular vesicles

doi: 10.1101/2024.04.03.587267

Figure Lengend Snippet: Development and validation of EV immuno-isolation method and application to NRXN3. A. Schematic of immuno-isolation of EVs enriched by ultracentrifugation from the cell culture media of K562 cells. B. Western blotting of CD81 after immuno-isolation of EVs. Equal amounts of EVs obtained by differential ultracentrifugation of K562 cell culture media were loaded as input or immuno-isolated using either an anti-CD81 antibody or a control non-specific antibody. PD = pulldown, FT = flow-through C. Schematic of EV immuno-isolation experiment from human plasma or CSF for western blotting analysis. EVs are immuno-isolated from biofluid without initial EV purification. Immuno-isolation was performed using a target antibody or control antibody (PD1 = pulldown 1) and the flow-through is subjected to a second immuno-isolation with the target antibody (PD2 = pulldown. 2) D. Western blots of CD9 (top), CD63 (middle), or CD81 (bottom) after immuno-isolation from human CSF. In each case, the first pulldown (PD1) was performed with antibodies against the target protein or a control antibody, and the second pulldown (PD2) was performed with antibodies against the target protein using the flow-through of PD1. E. Western blots of CD9 (top), CD63 (middle), or CD81 (bottom) after immuno-isolation from human plasma. In each case, the first pulldown (PD1) was performed with antibodies against the target protein or a control antibody, and the second pulldown (PD2) was performed with antibodies against the target protein using the flow-through of PD1. F. Schematic of EV immuno-isolation experiment from human plasma or EVs enriched by SEC from human plasma for assessment by Simoa in the flow-through (FT). G. Simoa measurements of CD9 (left), CD63 (middle), or CD81 (right) of the flow-through of human plasma after immuno-isolation with antibodies against CD9, CD63, CD81 or GFP (non-specific control). H. Simoa measurements of CD9 (left), CD63 (middle), or CD81 (right) of the flow-through of EVs enriched from human plasma by SEC after immuno-isolation with antibodies against CD9, CD63, CD81 or GFP (non-specific control). For G and H, two separate replicates of all samples were used and Simoa measurements were taken in duplicate for each sample. Error bars indicate SD. I. Schematic of NRXN3 immuno-isolation experiment. NRXN3 in pulldown fraction (PD) was measured using Western blotting. NRXN3 in flow-through (FT) was measured using Simoa. Immuno-isolation experiment was performed using antibody against target (NRXN3) or control (GFP or isotype control) J. Western blotting of NRXN3 after pulldown from CSF using either NRXN3 antibody or control antibody (GFP or isotype control). Simoa measurement of NRXN3 in flow-through after pulldown from CSF using either NRXN3 antibody or control antibody (GFP or isotype control). K. Western blotting of NRXN3 after pulldown from plasma using either NRXN3 antibody or control antibody (GFP or isotype control). Simoa measurement of NRXN3 in flow-through after pulldown from plasma using either NRXN3 antibody or control antibody (GFP or isotype control). For J and K, Simoa measurements were taken in duplicate for each sample. Error bars indicate SD.

Article Snippet: The following antibodies were used for immuno-isolation of NRXN3 and controls: sheep polyclonal NRXN3 (cat. AF5269, R&D Systems), GFP (4745-1051, Rockland), and Isotype Control (AB37385, Abcam).

Techniques: Biomarker Discovery, Isolation, Cell Culture, Western Blot, Control, Clinical Proteomics, Purification

Journal: Annals of neurology

Article Title: Mechanisms of Caspr2 antibodies in autoimmune encephalitis and neuromyotonia

doi: 10.1002/ana.25120

Figure Lengend Snippet: Clinical characteristics and antibody titers of Caspr2 positive subjects

Article Snippet: Wells were incubated with either anti-Caspr2 (R&D cat. # AF5245,1:100) or anti-contactin-2 (R&D cat. # AF1714, 1:1000) antibody for one hour followed by incubation with peroxidase-conjugated secondary antibody for 45 minutes.

Techniques:

Journal: Annals of neurology

Article Title: Mechanisms of Caspr2 antibodies in autoimmune encephalitis and neuromyotonia

doi: 10.1002/ana.25120

Figure Lengend Snippet: IgG subtypes of Caspr2 autoantibodies by ELISA

Article Snippet: Wells were incubated with either anti-Caspr2 (R&D cat. # AF5245,1:100) or anti-contactin-2 (R&D cat. # AF1714, 1:1000) antibody for one hour followed by incubation with peroxidase-conjugated secondary antibody for 45 minutes.

Techniques:

Journal: Ecotoxicology and environmental safety

Article Title: Exposure to polystyrene nanoplastics induces hepatotoxicity involving NRF2-NLRP3 signaling pathway in mice.

doi: 10.1016/j.ecoenv.2024.116439

Figure Lengend Snippet: Fig. 3. Effects of PS-NPs on expression of KEAP1 (A and B) and NRF2 (A and C) and levels of MDA (D), GSH (E), and CAT (F) in the liver of mice. *P < 0.05 and **P < 0.01, versus control group.

Article Snippet: The membranes were treated with specific antibodies antibodies raised against nuclear factor erythroid 2-related factor 2 (NRF2) (1:1000, Affinity, China; Cat. No. AF0639), p-NRF2 (1:1000, Affinity, China; Cat. No. DF7519), KEAP1 (1:1000, Affinity, China; Cat. No. AF5266), HO-1 (1:1000, Proteintech, China; Cat. No. 10701–1-AP), NQO1 (1:1000, Proteintech, China; Cat. No. 11451–1-AP), NLRP3 (1:1000, Abmart, China; Cat. No. M035175), caspase-1 (1:1000, Proteintech, China; Cat. No. 22915–1-AP), p-p65 (1:1000, Affinity, China; Cat. No. AF2006), p65 (1:1000, Affinity, China; Cat. No. AF5006), IL-6 (1:1000, Abmart, China; Cat. No. TD6087), IL-1β (1:1000, Abmart Medical Technologies, China; Cat. No. TA5103), and GAPDH (1:15000, Proteintech, China; Cat. No. 10494–1-AP) at 4°C overnight.

Techniques: Expressing, Control

Journal: Frontiers in Pharmacology

Article Title: Dehydrocostus Lactone Suppresses Dextran Sulfate Sodium-Induced Colitis by Targeting the IKKα/β-NF-κB and Keap1-Nrf2 Signalling Pathways

doi: 10.3389/fphar.2022.817596

Figure Lengend Snippet: DCL enhances the activation of Keap1/Nrf2/HO-1 in LPS/IFNγ-stimulated RAW264.7 macrophages. RAW264.7 cells were treated with DCL (1, 3, and 9 μM) or curcumin (5 μM) for 1 h, and followed by LPS/IFNγ incubation for additional 8 h. The protein expression of Keap1 (A) , Nrf2 (C) , and HO-1 (G) were measured by western blot. The densitometry analysis of Keap1 (B) , Nrf2 (D) , and HO-1 (H) , normalized against GAPDH or α-Tubulin, respectively. The distribution of Nrf2 in cytoplasmic and nuclear fractions was analyzed using western blotting (E) . The densitometry analysis of cytoplasmic and nuclear Nrf2, normalized against GAPDH and PARP1, respectively (F) . (I , J) ROS production was measured using flow cytometry and relative statistical analysis. Data are represented as mean ± SEM, n = 3. # p < 0.05, ## p < 0.01, ### p < 0.001, #### p < 0.0001 compared to CK group. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 compared to LPS/IFNγ group.

Article Snippet: Primary antibodies against Keap1 (Cat# AF5266), Nrf2 (Cat# AF7006) and HO-1 (Cat# AF5393) were purchased from Affinity Biosciences (Cincinnati, OH, United States).

Techniques: Activation Assay, Incubation, Expressing, Western Blot, Flow Cytometry

Journal: Frontiers in Pharmacology

Article Title: Dehydrocostus Lactone Suppresses Dextran Sulfate Sodium-Induced Colitis by Targeting the IKKα/β-NF-κB and Keap1-Nrf2 Signalling Pathways

doi: 10.3389/fphar.2022.817596

Figure Lengend Snippet: DCL directly interacts with IKKα/β and Keap1 respectively. The effects of DCL on the thermal stability of IKKα/β ( A , upper panel) and Keap1 ( D , upper panel) were analyzed by CETSA coupling western blotting assay. The CETSA curves of IKKα/β ( A , bottom panel) and Keap1 ( D , bottom panel) were shown. The densitometries of IKKα/β and Keap1 were normalized with respect to those obtained at 45°C. The effects of DCL on the protein’s proteolytic susceptibility of IKKα/β ( B , upper panel) and Keap1 ( E , upper panel) were analyzed by DARTS assay. The densitometry analysis of IKKα/β ( B , bottom panel) and Keap1 ( E , bottom panel) were shown. Molecular docking of DCL with IKKβ ( C , PDB: 3QA8) and Keap1 ( F , PDB: 4CXI). The covalent bonds are shown in purple. Data are represented as mean ± SEM, n = 3. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Primary antibodies against Keap1 (Cat# AF5266), Nrf2 (Cat# AF7006) and HO-1 (Cat# AF5393) were purchased from Affinity Biosciences (Cincinnati, OH, United States).

Techniques: Western Blot