Journal: Frontiers in aging neuroscience

Article Title: Adenosine A 2A Receptor in Bone Marrow-Derived Cells Mediated Macrophages M2 Polarization via PPARγ-P65 Pathway in Chronic Hypoperfusion Situation.

doi: 10.3389/fnagi.2021.792733

Figure Lengend Snippet: FIGURE 5 | The expression of PPARγ, P65, and p-P65 was altered in macrophages cultured in low glucose and hypoxia. (A) Representative electrophoretic bands showing the expression of PPARγ, P65 and p-P65 in control, CGS21680 and SCH58261 treated macrophages at post-culture. β-actin was used as internal control. (B) Statistical analysis showing the expression of PPARγ in macrophages was increased gradually in low glucose and hypoxic conditions, and was increased further by CGS21680 at post-culture 12 h, but SCH58261 reduced the expression of PPARγ at post-culture (ns: P > 0.05, ***P < 0.001. Two-way ANOVA test followed by Turkey’s multiple comparisons test. n = 3 independent experiments for each group). (C) Statistical analysis showing the expression of P65 was increased in macrophages at post-culture 12 and 24 h, and the expression was further increased by CGS21680 at 12 h and SCH58261 at 6 and 12 h (ns: P > 0.05, ***P < 0.001. Two-way ANOVA test followed by Turkey’s multiple comparisons test. n = 3 independent experiments for each group). (D) Statistical analysis showing the expression of p-P65 was increased by CGS21680 at post-culture 6 and 24 h, and was increased in SCH58261 treated macrophages at post-culture 6, 12, and 24 h (*P < 0.05, ***P < 0.001. Two-way ANOVA test followed by Turkey’s multiple comparisons test. n = 3 independent experiments for each group). (E,F) Statistical analysis showing the mRNA of PPARγ was increased in CGS21680 treated macrophages at 2 and 12 h, but not affected by SCH58261. The expression of P65 mRNA was increased in SCH58261 treated macrophages at post-culture 12 h (ns: P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001. Two-way ANOVA test followed by Turkey’s multiple comparisons test. n = 3 independent experiments for each group). ns, non significant; p >0.05.

Article Snippet: The membranes were blocked by 5% skimmed milk and incubated overnight at 4◦C with the following primary antibodies: a rabbit anti-PPARγ antibody (1:1000, AF6284, Affinity Bioscience, Beijing, China), rabbit anti-P65 antibody (1:1000, NB100-2176, NOVUS Biologicals, Building IV Centennial, CO 80112, USA), or rabbit anti-p-P65 antibody (1:1000, NB100-82088, NOVUS Biologicals, Building IV Centennial, CO 80112, USA).

Techniques: Expressing, Cell Culture, Control

Journal: Frontiers in aging neuroscience

Article Title: Adenosine A 2A Receptor in Bone Marrow-Derived Cells Mediated Macrophages M2 Polarization via PPARγ-P65 Pathway in Chronic Hypoperfusion Situation.

doi: 10.3389/fnagi.2021.792733

Figure Lengend Snippet: FIGURE 7 | PPARγ regulated the expression of P65 and p-P65 in CGS21680 treated macrophages. (A) Representative electrophoretic bands showing the expression of P65 and p-P65 in PPARγ shRNA pre-transfected macrophages with or without CGS21680 treatment after cultured in low glucose and hypoxic conditions. (B,C) Densitometric analysis results showing the expression of P65 were reduced in shRNA targeted macrophages and further reduced by CGS21680 at post-culture 12 h (*P < 0.05, ***P < 0.001. Two-way ANOVA test followed by Turkey’s multiple comparisons test. n = 3 independent experiments for each group). (C) The ratio of p-P65/P65 was increased in macrophages upon PPARγ knockdown at post-culture 12 and 24 h, but was reduced in by additional CGS21680 treatment at post-culture 2 and 12 h (***P < 0.001. Two-way ANOVA test followed by Turkey’s multiple comparisons test. n = 3 independent experiments for each group). (D) RT-PCR results showing the mRNA expression of P65 was reduced in macrophages upon PPARγ knockdown at post-culture 12 h and was potentiated by additional CGS21680 treatment at post-culture 6 and 12 h (*P < 0.05, ***P < 0.001. Two-way ANOVA test followed by Turkey’s multiple comparisons test. n = 3 independent experiments for each group). **P < 0.01.

Article Snippet: The membranes were blocked by 5% skimmed milk and incubated overnight at 4◦C with the following primary antibodies: a rabbit anti-PPARγ antibody (1:1000, AF6284, Affinity Bioscience, Beijing, China), rabbit anti-P65 antibody (1:1000, NB100-2176, NOVUS Biologicals, Building IV Centennial, CO 80112, USA), or rabbit anti-p-P65 antibody (1:1000, NB100-82088, NOVUS Biologicals, Building IV Centennial, CO 80112, USA).

Techniques: Expressing, shRNA, Transfection, Cell Culture, Knockdown, Reverse Transcription Polymerase Chain Reaction

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: 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: 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