Journal: Cell reports

Article Title: The IRAK1/IRF5 axis initiates IL-12 response by dendritic cells and control of Toxoplasma gondii infection

doi: 10.1016/j.celrep.2024.113795

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: The following antibodies and dilutions were used: IRAK1 (Cell Signaling, 4504S, 1:1000), IRAK2 (Abcam, ab62419, 1:500), IRAK4 (Novus, NB500–597, 1:1000), MyD88 (R&D Systems, AF3109, 1:500), rodent-specific IRF5 (Cell Signaling, 4950, 1:1000), USF2 (Novus Biologicals, NBP1–92649, 1:2000), c-Rel (Cell Signaling, 67489, 1:1000), phospho-IκB-α (Ser32) (Cell Signaling, 2859, 1:750), IκB-α (Cell Signaling, 4812S, 1:1000), phospho-RelA/NF-κB (Ser 536) (Cell Signaling, 3033, 1:1000), RelA/NF-κB (Novus Biologicals, NB100–2176, 1:1000), Actin (Sigma-Aldrich, A2066, 1:3000), phospho-SAPK/JNK (Thr 183/Tyr 185) (Cell Signaling, 4668T, 1:1000), phospho-p38 MAPK (Thr 180/Tyr 182) (Cell Signaling, 4511T, 1:1000), phospho-p44/42 MAPK (Erk 1/2) (Thr 202/Tyr 204) (Cell Signaling, 4370T, 1:2000), phosphor-IKK (Ser 176/180) (Cell Signaling, 2697, 1:1000), CNBP (Santa Cruz, sc-515387-X, 1:200), anti-Goat-IgG HRP-conjugated (R&D Systems, HAF017, 1:5000), anti-Rabbit-IgG HRP-conjugated (Sigma-Aldrich, A0545, 1:5000), anti-Mouse-IgG HRP-conjugated (Cell Signaling, 7076S, 1:5000).

Techniques: Recombinant, Modification, Saline, Protease Inhibitor, Marker, Western Blot, Extraction, Enzyme-linked Immunosorbent Assay, Software

Journal: Evidence-based complementary and alternative medicine : eCAM

Article Title: Zanthoxylum bungeanum Seed Oil Attenuates LPS-Induced BEAS-2B Cell Activation and Inflammation by Inhibiting the TLR4/MyD88/NF- κ B Signaling Pathway.

doi: 10.1155/2021/2073296

Figure Lengend Snippet: Figure 5: Docking results for the active components in ZBSO with key protein molecules in the TLR4/MyD88/NF-κB signaling pathway. (a) ,e structures of active components in ZBSO were obtained by screening. (b) α-Linolenic acid, quercetin, and TLR4, MyD88, or NF- κB p65 proteins in molecular docking mode.

Article Snippet: LPS was obtained from Solarbio (055: B5, Beijing, China).,e antibodies against MyD88 (23230-1-AP), NF-κB p65 (10745-1-AP), and phospho-NF-κB p65 were obtained from Proteintech (Hubei, China).,e antibodies against TRL4 (GB11519) and β-actin were gained from Servicebio (Hubei, China).

Techniques:

Journal: Evidence-based complementary and alternative medicine : eCAM

Article Title: Zanthoxylum bungeanum Seed Oil Attenuates LPS-Induced BEAS-2B Cell Activation and Inflammation by Inhibiting the TLR4/MyD88/NF- κ B Signaling Pathway.

doi: 10.1155/2021/2073296

Figure Lengend Snippet: Figure 6: ZBSO inhibition of LPS-induced p65 nuclear translocation. (a) Typical immunofluorescence images of NF-κB p65 (red) and Hoechst 33258 (blue) induced by LPS (20 × magnification). Western blot analysis of NF-κB p65 total protein, NF-κB p65 phosphorylation (b), and NF-κB p65 in the nucleus (c) and cytoplasm (d). ,e values are expressed as the mean ± SD (n 3) of three independent ex- periments. #p < 0.05 and ##p < 0.01 versus the control group; ∗p < 0.05 and ∗∗p < 0.01 versus the LPS group.

Article Snippet: LPS was obtained from Solarbio (055: B5, Beijing, China).,e antibodies against MyD88 (23230-1-AP), NF-κB p65 (10745-1-AP), and phospho-NF-κB p65 were obtained from Proteintech (Hubei, China).,e antibodies against TRL4 (GB11519) and β-actin were gained from Servicebio (Hubei, China).

Techniques: Inhibition, Translocation Assay, Western Blot, Phospho-proteomics, Control

Journal: Cells

Article Title: Gas6/TAM Signalling Negatively Regulates Inflammatory Induction of GM-CSF in Mouse Brain Microglia

doi: 10.3390/cells10123281

Figure Lengend Snippet: Gas6 inhibits LPS-induced nuclear translocation of the NF-κB p65 subunit in microglia. Pure microglial cell cultures were treated with LPS (10 ng/mL) for 30 min with or without 1 h pre-incubation with Gas6 (1.6 µg/mL), which then remained throughout. ( A ) Cells underwent immunofluorescence staining with anti-p65 primary antibody with AlexaFluor647 anti-mouse secondary antibody and DAPI nuclear counterstaining. Scale bar = 100 µm. ( B ) p65 staining within the nuclear area of each cell was quantified for each treatment group. Data is shown in a violin plot with median and interquartile ranges visible ( n > 30 cells). Data was statistically analysed using one-way ANOVA; **** p < 0.0001 vs. both other conditions.

Article Snippet: For staining, coverslips were incubated in primary mouse anti-p65 antibody (1:300; Proteintech, Manchester, UK) at 4 °C overnight.

Techniques: Translocation Assay, Incubation, Immunofluorescence, Staining

Journal: The Prostate

Article Title: RELA is sufficient to mediate interleukin-1 repression of androgen receptor expression and activity in an LNCaP disease progression model

doi: 10.1002/pros.23925

Figure Lengend Snippet: RELA mediates IL-1 repression of AR. A, RT-qPCR and B, Western blot analysis and densitometry were performed for LNCaP, C4-2, and C4-2B cells transfected with 70 nM of nontargeting or RELA siRNA (Dharmacon) for 1 day followed by treatment with vehicle control (Veh), IL-1α, IL-1β for 2 days. LNCaP cells were treated with 0.5 ng/mL IL-1, and C4-2 and C4-2B cells were treated with 25 ng/mL IL-1. RELA siRNA attenuates IL-1 downregulation of AR and PSA mRNA and protein, and attenuates IL-1 upregulation of SOD2 mRNA and protein. mRNA fold change is normalized to the vehicle control. Error bars indicate ±SD of three biological replicates; *P ≤ .05, **P ≤ .005, ***P ≤ .0005. AR, androgen receptor; IL, interleukin; mRNA, messenger RNA; PSA, prostate-specific antigen; RT-qPCR, reverse-transcription quantitative polymerase chain reaction; SD, standard deviation; siRNA, small interfering RNA; SOD2, superoxide dismutase 2

Article Snippet: The following siRNA concentrations were used: 70 nM nontargeting siRNA (D-001206-13-05; Dharmacon), RELA siRNA (M-003533-02-0005; Dharmacon), NFKB1 siRNA (M-003520-01-0005; Dharmacon), or p62 siRNA (M-010230-00-0005; Dharmacon); 100 nM nontargeting siRNA, RELB siRNA (M-004767-02-0005; Dharmacon), c-REL siRNA (M-004768-01-0005; Dharmacon), or NFKB2 siRNA (M-003918-02–0005; Dharmacon); 70 nM nontargeting siRNA (SR30004; Origene) or RELA siRNA (SR321602; Origene).

Techniques: Quantitative RT-PCR, Western Blot, Transfection, Real-time Polymerase Chain Reaction, Standard Deviation, Small Interfering RNA

Journal: The Prostate

Article Title: RELA is sufficient to mediate interleukin-1 repression of androgen receptor expression and activity in an LNCaP disease progression model

doi: 10.1002/pros.23925

Figure Lengend Snippet: RELA siRNA restores nuclear AR accumulation in IL-1-treated PCa cells. C4-2 and C4-2B cells transfected with 70 nM of nontargeting or RELA siRNA (Dharmacon) for 1 day followed by treatment with vehicle control (Veh), 25 ng/mL IL-1α, or 25 ng/mL IL-1β for 2 days. A, Cells were fixed and immunostained for AR (Texas Red) and cell nuclei (DAPI, blue) and imaged at ×20 magnification. AR/DAPI merge images are shown for C4-2 (left) and C4-2B (right). B, The ratio (AR/DAPI) of the number of cells accumulating AR to the number of DAPI-stained cells was calculated for five microscopy fields at ×10 magnification, n > 300 cells per field. AR/DAPI ratio was normalized to vehicle control siRNA. RELA siRNA restores AR nuclear accumulation in IL-1-treated cells. Error bars indicate ±SD of five microscopy fields; ***P ≤ .0005. AR, androgen receptor; DAPI, 4′,6-diamidino-2-phenylindole; IL, interleukin; SD, standard deviation; siRNA, small interfering RNA [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: The following siRNA concentrations were used: 70 nM nontargeting siRNA (D-001206-13-05; Dharmacon), RELA siRNA (M-003533-02-0005; Dharmacon), NFKB1 siRNA (M-003520-01-0005; Dharmacon), or p62 siRNA (M-010230-00-0005; Dharmacon); 100 nM nontargeting siRNA, RELB siRNA (M-004767-02-0005; Dharmacon), c-REL siRNA (M-004768-01-0005; Dharmacon), or NFKB2 siRNA (M-003918-02–0005; Dharmacon); 70 nM nontargeting siRNA (SR30004; Origene) or RELA siRNA (SR321602; Origene).

Techniques: Transfection, Staining, Microscopy, Standard Deviation, Small Interfering RNA

Journal: Molecular Medicine Reports

Article Title: Heme oxygenase 1‑overexpressing bone marrow mesenchymal stem cell‑derived exosomes suppress interleukin‑1 beta‑induced apoptosis and aging of nucleus pulposus cells

doi: 10.3892/mmr.2025.13481

Figure Lengend Snippet: Activation of the NF-κB signalling in NP cells induced by IL-1β was repressed after Exo-HO-1 treatment. Western blotting was performed to detect p-p65 and/or p65 protein levels in NP cells with different treatments (control, IL-1β, IL-1β + Exo and IL-1β + Exo-HO-1) as well as in the nuclear (N) and cytoplasmic (C) fractions of the cells. Histone H3 was used as an internal reference for the nucleus. ***P<0.001 vs. control; # P<0.05, ## P<0.01 and ### P<0.001 vs. IL-1β. NP, nucleus pulposus; IL, interleukin; Exo, exosomes; HO-1, heme oxygenase 1; p-phosphorylated.

Article Snippet: After blocking with a rapid blocking solution (Beyotime Institute of Biotechnology) at 4°C overnight, the membranes were incubated with appropriate primary antibodies including anti-HO-1 (cat. no. GTX637432; 1:2,000; GeneTex, Inc.), anti-TGS101 (cat. no. A01233-2, 0.25 μg/ml; Boster Bio), anti-CD63 (FNab01490; 1:1,000; Fine Biotech Co., Ltd, Wuhan, China) and anti-Cainexin (cat. no. GTX109669; 1:5,000; GeneTex, Inc.), Bcl-2 (cat. no. FNab00839; 1:1,000; Wuhan Fine Biotech Co., Ltd.), Bax (cat. no. FNab00810; 1:2,000; Wuhan Fine Biotech Co., Ltd.), cleaved caspase 3 (cat. no. MBS9410752; 1:1,000; MyBioSource, Inc.), anti-p65 (cat. no. PB9324; 0.5 μg/ml; Boster Bio), anti-p-p65 (cat. no. A00284S468-2; 1:2,000; Boster Bio), anti-GAPDH (cat. no. H00227; 1:5,000; Boster Bio) and anti-Histone H3 (cat. no. M12477-9; 1:1,000; Boster Bio) antibodies.

Techniques: Activation Assay, Western Blot, Control

Journal: BMC Biology

Article Title: Temperature-induced embryonic diapause in chickens is mediated by PKC-NF-κB-IRF1 signaling

doi: 10.1186/s12915-023-01550-0

Figure Lengend Snippet: Rapid induction of IRF1 transcription is mediated by PKC-NF-kB signaling. A PKC activity levels in chicken embryos treated with cold stimulation (28°C) for 12 hours, N = 7. B PKC activity levels in cESCs treated with cold stimulation (28°C) for 3 hours, N = 7. C Immunostaining of cESCs with or without 3 hours of cold stimuli. Staining was performed with an antibody against PKC (green). DAPI was applied as a counterstain (blue). Scale: 20 μm. D NF-kB luciferase levels in cESCs treated with cold stimulation (28°C), a PKC activator (TPA) or a PKC inhibitor (GO 6983) for 3 hours. cESCs transfected with an NF-kB luciferase reporter vector and untransfected cESCs served as the control group (Ctrl) and negative control group (NC), respectively, N = 3. E Immunostaining of cESCs with or without 3 hours of cold stimuli. Staining was performed with an antibody against P65 (green) or DAPI (blue). Scale: 20 μm. F Changes in gene expression after treatment with cold stimuli, and an activator and/or inhibitor of PKC and NF-kB for 3 hours in cESCs, N = 4. The mean ± SEM is shown in all panels. * p < 0.05; ** p < 0.01; *** p < 0.001. N indicates biological replicates

Article Snippet: The sections were incubated overnight at 4°C with primary antibodies against H3S10P (Cat: 3377T; CST; 1:200), PCNA (Cat: abs120180; Absin, Shanghai, China; 1:200), PKC (Cat: NB600-201; Novus, Littleton, CO, USA; 1:100) or P65 (Cat: NBP2-24541; Novus; 1:100).

Techniques: Activity Assay, Immunostaining, Staining, Luciferase, Transfection, Plasmid Preparation, Control, Negative Control, Expressing