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: 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: Cancer Research

Article Title: Nucleolar Targeting of RelA(p65) Is Regulated by COMMD1-Dependent Ubiquitination

doi: 10.1158/0008-5472.can-09-1397

Figure Lengend Snippet: Figure 1. Nucleolar translocation of RelA and proteasome activity. A, SW480 colon cancer cells were treated with aspirin (0–5 mmol/L, 16 h). 20S proteasome activity was measured in whole-cell lysates using fluorometric assay. Columns, percentage of basal (0 mmol/L) activity (n = 4); bars, SE. B, SW480 cells were treated with 10 mmol/L aspirin for the times specified and then 20S proteasome activity was measured as above. Columns, percentage of basal (0 h) activity (n = 4); bars, SE. C, SW480 cells were transfected with HA-tagged ubiquitin 24 h before treatment with aspirin (10 mmol/L) for the specified times. Anti-HA Western blot analysis was done on whole-cell extracts. Actin is used as a protein loading control. D, SW480 cells treated with aspirin (0 or 5 mmol/L, 16 h) were fractionated using sucrose gradients. 20S proteasome activity was measured in lysates from the nuclear and nucleolar fractions as above. Columns, percentage of nontreated (NT) nuclear proteasome activity (n = 3); bars, SE. Inset, anti-C23, fibrillarin (nucleolar markers), and lamin B (nonnucleolar protein) Western blot analysis done on lysates from the nuclear and nucleolar fractions.

Article Snippet: The following primary antibodies were used: p65(C-20), p65(F-6), nucleolin (C23), fibrillarin, glutathione S-transferase (GST), and green fluorescent protein (GFP; all from Santa Cruz); His [generated in-house (purified form)]; ubiquitin (rabbit antibody; Dako Cytomation); ubiquitin (mouse monoclonal; Stressgen); filamin A (Bethyl Laboratories); phospho-p65 (Ser468) and phospho-p65 (Ser536) (Cell Signaling Technology).

Techniques: Translocation Assay, Activity Assay, Transfection, Ubiquitin Proteomics, Western Blot, Control

Journal: Cancer Research

Article Title: Nucleolar Targeting of RelA(p65) Is Regulated by COMMD1-Dependent Ubiquitination

doi: 10.1158/0008-5472.can-09-1397

Figure Lengend Snippet: Figure 2. Ubiquitination of RelA precedes nucleolar translocation of the protein. A, SW480 cells were treated with aspirin (0–5 mmol/L, 16 h). Top, the levels of high molecular weight [indicative of ubiquitination (Ub.)] and native RelA were determined by Western blot analysis of whole-cell lysates. Bottom, RelA was immunoprecipitated (IP) from lysates, and recovered protein analyzed using anti-ubiquitin Western blot analysis (WB Ub.). Immunoprecipitation with isotyped IgG controlled for specificity. Stripped gels were reprobed for RelA to monitor the amount of RelA immunoprecipitated (WB. RelA). B, SW480 cells were transfected with WT 6His-ubiquitin (ub) or the R7 mutant that cannot form polyubiquitin chains. Twenty-four hours after transfection, cells were either left untreated (NT) or treated with aspirin (5 mmol/L) or MG132 (25 μmol/L) for a further 16 h. His-tagged proteins were precipitated from whole-cell lysates using Ni-agarose beads and then subjected to anti-RelA and anti-histidine (his) Western blot analysis (WB). NS, nonspecific band. C, SW480 cells were treated with aspirin (10 mmol/L) for the times specified. Top, the levels of native and high molecular weight forms of RelA were determined using Western blot analysis. Bottom, the nuclear distribution of RelA was assessed by immunocytochemical staining (magnification, ×63). D, SW480 cells were exposed to UV-C radiation (40 J/m2) or TNF (10 ng/mL) for the times specified. Top, anti-RelA Western blot analysis done on whole-cell lysates. Bottom, immunomicrographs showing the cellular distribution of RelA (magnification, ×63).

Article Snippet: The following primary antibodies were used: p65(C-20), p65(F-6), nucleolin (C23), fibrillarin, glutathione S-transferase (GST), and green fluorescent protein (GFP; all from Santa Cruz); His [generated in-house (purified form)]; ubiquitin (rabbit antibody; Dako Cytomation); ubiquitin (mouse monoclonal; Stressgen); filamin A (Bethyl Laboratories); phospho-p65 (Ser468) and phospho-p65 (Ser536) (Cell Signaling Technology).

Techniques: Ubiquitin Proteomics, Translocation Assay, High Molecular Weight, Western Blot, Immunoprecipitation, Transfection, Mutagenesis, Staining

Journal: Cancer Research

Article Title: Nucleolar Targeting of RelA(p65) Is Regulated by COMMD1-Dependent Ubiquitination

doi: 10.1158/0008-5472.can-09-1397

Figure Lengend Snippet: Figure 3. Proteasome inhibition induces ubiquitination and nucleolar translocation of RelA. A, SW480 cells were treated with carrier (DMSO), MG132 (25 μmol/L), or lactacystin (Lact.; 35 μmol/L) for 16 h. Immunomicrographs (×63) show the cellular localization of RelA. C23 staining identifies nucleoli. DNA is stained by DAPI. The percentage of cells showing nucleolar RelA (as indicated by colocalization with C23) was determined in at least 200 cells from at least five randomly selected fields of view (n = 3). B and D, SW480 cells were treated with MG132 (25 μmol/L) or lactacystin (50 μmol/L) for the times specified. Top, anti-RelA Western blot analysis showing native and high molecular weight forms of the protein, indicative of the addition of multiple ubiquitin chains (Ub. RelA). Middle, the cellular distribution of RelA was determined by immunocytochemical staining (magnification, ×63). Bottom, for each time point, the percentage of cells showing nucleolar localization of RelA (as depicted by areas devoid of DAPI staining) was determined as in A (n = 3). B, bottom, gray line, SW480 cells were transiently transfected with the NF-κB–dependent 3×κB ConA-luc and the control pCMVβ reporter constructs. Twenty-four hours after transfection, cells were treated with MG132 (25 μmol/L) for the times specified. Results were normalized using β-galactosidase activity and are presented as the percentage of relative luciferase activity compared with basal (carrier treated) controls (n = 2); bars, SD. C, SW480 cells were transfected with GFP-RelA 24 h before treatment with MG132 (25 μmol/L, 0 or 2 h). Immunoprecipitation (IP) of ubiquitinated proteins followed by anti-RelA Western blot analysis (WB) of recovered complexes confirmed an increase in ubiquitinated forms of RelA 2 h after MG132 treatment. Control immunoprecipitations were done with preimmune (IgG) serum. RelA in input samples is shown.

Article Snippet: The following primary antibodies were used: p65(C-20), p65(F-6), nucleolin (C23), fibrillarin, glutathione S-transferase (GST), and green fluorescent protein (GFP; all from Santa Cruz); His [generated in-house (purified form)]; ubiquitin (rabbit antibody; Dako Cytomation); ubiquitin (mouse monoclonal; Stressgen); filamin A (Bethyl Laboratories); phospho-p65 (Ser468) and phospho-p65 (Ser536) (Cell Signaling Technology).

Techniques: Inhibition, Ubiquitin Proteomics, Translocation Assay, Staining, Western Blot, High Molecular Weight, Transfection, Control, Construct, Activity Assay, Luciferase, Immunoprecipitation

Journal: Cancer Research

Article Title: Nucleolar Targeting of RelA(p65) Is Regulated by COMMD1-Dependent Ubiquitination

doi: 10.1158/0008-5472.can-09-1397

Figure Lengend Snippet: Figure 4. Amino acids 27–30 are required for ubiquitination and nucleolar translocation of RelA. A, SW480 cells were transfected with the specified plasmids and treated with MG132 (25 μmol/L) or lactacystin (35 μmol/L) for 16 h as above; then the cellular localization of GFP-tagged RelA was determined in live cells using an Axiovert 100 inverted fluorescent microscope (×40; left). Nucleoli are indicated by arrows in phase-contrast images. Right images, fixed cells counterstained with DAPI (DNA stain). Nucleoli are depicted by areas devoid of DAPI staining (magnification, ×63). B, SW480 cells were transfected with the specified GFP plasmids, then either left untreated (Asp. −) or treated with aspirin (10 mol/L), carrier (MG132 −), or MG132 (25 μmol/L) for 2 h. The levels of native and high molecular weight (Ub) RelA and total protein ubiquitination were determined by Western blot analysis. C, SW480 cells were transfected and treated with MG132 (25 μmol/L) or aspirin (10 mmol/L) as above. The levels of ubiquitinated RelA were determined by anti-ubiquitin immunoprecipitation (IP) followed by anti-RelA Western blot analysis (WB). Membranes were stripped and reprobed to show the effects of the agents on total protein ubiquitination. Right, SW480 cells constitutively expressing GFP-RelA WT or Δ27–30 were transfected with 6His-ubiquitin. Cells were then treated for 2 h with aspirin (10 mmol/L), and ubiquitinated proteins precipitated using Ni-agarose beads. Precipitated proteins were subjected to anti-RelA and anti-His Western blot analysis. D, SW480 cells were transfected with the specified vectors and then treated with MG132 (25 μmol/L) or lactacystin (0–75 μmol/L) for 16 h. Annexin V-biotin staining, with a Texas red-streptavidin conjugate, was used to identify apoptotic cells. The percentage of cells expressing GFP-tagged RelA undergoing apoptosis was determined by fluorescent microscopy in at least 250 transfected cells for each sample. Columns, mean of at least three independent experiments; bars, SE.

Article Snippet: The following primary antibodies were used: p65(C-20), p65(F-6), nucleolin (C23), fibrillarin, glutathione S-transferase (GST), and green fluorescent protein (GFP; all from Santa Cruz); His [generated in-house (purified form)]; ubiquitin (rabbit antibody; Dako Cytomation); ubiquitin (mouse monoclonal; Stressgen); filamin A (Bethyl Laboratories); phospho-p65 (Ser468) and phospho-p65 (Ser536) (Cell Signaling Technology).

Techniques: Ubiquitin Proteomics, Translocation Assay, Transfection, Microscopy, Staining, High Molecular Weight, Western Blot, Immunoprecipitation, Expressing

Journal: Cancer Research

Article Title: Nucleolar Targeting of RelA(p65) Is Regulated by COMMD1-Dependent Ubiquitination

doi: 10.1158/0008-5472.can-09-1397

Figure Lengend Snippet: Figure 5. COMMD1 modulates the nuclear distribution of RelA. A, immunomicrographs (magnification, ×63) showing aspirin (5 mmol/L, 16 h)-mediated nucleolar translocation of RelA in SW480 cells transfected with GST-COMMD1 or control (GST) vector. NT, nontreated. The percentage of cells in the total cell population showing nucleolar RelA (as indicated by colocalization with the nucleolar marker fibrillarin) was determined in at least 200 cells. Columns, mean (n = 4); bars, SE. Inset, anti-COMMD1 immunoblot showing levels of GST-COMMD1 in transfected SW480 cells. B and C, SW480 cells were transfected with control or COMMD1 siRNA (C1/1 and C1/2) and then treated with aspirin (10 mmol/L) for 0 to 8 h. B, anti-RelA immunoblot showing levels of native and ubiquitinylated RelA in response to aspirin (10 mmol/L, 2 h) treatment. Native RelA controls for protein loading. Numbers indicate the mean relative intensity (RI) of high molecular weight RelA (compared with native RelA; quantified using ImageJ; n = 3). C, immunocytochemical staining determined the nuclear distribution of RelA. The percentage of cells showing nucleolar RelA was calculated as above. Inset, Western blot analysis determined the levels of COMMD1 with (+) and without (−) aspirin (10 mmol/L, 2 h) treatment. Actin was used as a control for protein loading. D, SW480 cells were transfected as indicated, then either untreated or treated with aspirin (5 mmol/L) for 24 h. Apoptotic cells were visualized using fluorescent microscopy and the percentage of apoptotic cells within the total cell population was calculated. Columns, average fold increase in apoptosis in response to aspirin compared with the equivalent nontreated control (n = 3); bars, SE.

Article Snippet: The following primary antibodies were used: p65(C-20), p65(F-6), nucleolin (C23), fibrillarin, glutathione S-transferase (GST), and green fluorescent protein (GFP; all from Santa Cruz); His [generated in-house (purified form)]; ubiquitin (rabbit antibody; Dako Cytomation); ubiquitin (mouse monoclonal; Stressgen); filamin A (Bethyl Laboratories); phospho-p65 (Ser468) and phospho-p65 (Ser536) (Cell Signaling Technology).

Techniques: Translocation Assay, Transfection, Control, Plasmid Preparation, Marker, Western Blot, High Molecular Weight, Staining, Microscopy

Journal: Cancer Research

Article Title: Nucleolar Targeting of RelA(p65) Is Regulated by COMMD1-Dependent Ubiquitination

doi: 10.1158/0008-5472.can-09-1397

Figure Lengend Snippet: Figure 6. Aspirin modulates the COMMD1-RelA interaction. A, SW480 cells were treated with 10 mmol/L aspirin for the times indicated and COMMD1 protein levels determined by Western blot analysis. Actin was used as a control for protein loading. B, SW480 cells stably expressing GFP-RelA were transfected with either GST-COMMD1 or an empty vector control (Vec-GST). Twenty-four hours after transfection, cells were treated with 10 mmol/L aspirin for the indicated times, and GST-tagged proteins precipitated from whole-cell lysates using glutathione Sepharose (GSH beads). Anti-RelA Western blot analysis (WB) of precipitated proteins confirmed an interaction between COMMD1 and RelA in response to aspirin treatment. Gels were reprobed with GST to examine the levels of precipitated GST-COMMD1 and GST-control. GFP-RelA and GST-COMMD1 levels in input samples are shown. C, SW480 cells were treated with either aspirin (10 mmol/L) or MG132 (25 μmol/L) for 2 h in the presence or absence of calyculin A (50 nmol/L). Anti-RelA immunoblot shows native and ubiquitinated (Ub) protein in whole-cell extracts. The levels of phosphorylated RelA (S468 and S563) were also determined by Western blot analysis. Filamin was used as a control for protein loading. D, model for nucleolar translocation of RelA and NF-κB–regulated apoptosis in colorectal cancer cells. See Discussion for details.

Article Snippet: The following primary antibodies were used: p65(C-20), p65(F-6), nucleolin (C23), fibrillarin, glutathione S-transferase (GST), and green fluorescent protein (GFP; all from Santa Cruz); His [generated in-house (purified form)]; ubiquitin (rabbit antibody; Dako Cytomation); ubiquitin (mouse monoclonal; Stressgen); filamin A (Bethyl Laboratories); phospho-p65 (Ser468) and phospho-p65 (Ser536) (Cell Signaling Technology).

Techniques: Western Blot, Control, Stable Transfection, Expressing, Transfection, Plasmid Preparation, Translocation Assay

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

Journal: International Journal of Molecular Sciences

Article Title: Resveratrol Modulates Chemosensitisation to 5-FU via β1-Integrin/HIF-1α Axis in CRC Tumor Microenvironment

doi: 10.3390/ijms24054988

Figure Lengend Snippet: Resveratrol’s reduction of inflammation, vascularisation as well as cancer stemness and elevation of apoptosis via β1-integrin receptors in HCT-116/HCT-116R cells shown by Western blot analysis. X -axis: HCT-116 ( A ) and HCT-116R ( B ) cells in alginate drops were left untreated alone (Co.) or in TME, where they were left untreated or were treated with 2 nM 5-FU, 5 µM resveratrol, 0.5 µM β1-SO, 0.5 µM β1-ASO or combinations thereof. Samples were immunoblotted with antibodies against NF-kB (unphosphorylated NF-kB), p-NF-kB (phosphorylated NF-kB), cleaved-caspase-3, HIF-1α, VEGF, CD44, CD133, ALDH1 and β-actin (loading control). Y -axis shows densitometric units. Relative to TME control, values were p < 0.05 (⋆) and p < 0.01 (⋆⋆).

Article Snippet: The monoclonal antibodies against phospho-p65-NF-kB (#MAB7226), p65-NF-kB (#MAB5078) as well as polyclonal anti-cleaved-caspase-3 (#AF835) were acquired from R&D Systems (Heidelberg, Germany), while monoclonal antibody against β1-integrin (#14-0299-82) was from Thermo Fisher Scientific (Langenselbold, Germany).

Techniques: Western Blot, Control