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: Clinical, Cosmetic and Investigational Dermatology

Article Title: A Topical Chinese Herbal Alleviates Psoriasis by Regulating Keratinocytes Pyroptosis Through Inhibition of NLRP3 Inflammasome Activation

doi: 10.2147/CCID.S559712

Figure Lengend Snippet: ( A – C ) mRNA levels of NLRP3, Caspase-1, GSDMD in NHEK cells, n = 3. ( D – I ) Protein levels of NF-κB, P65, NLRP3, caspase-1, GSDMD in NHEK cells, n = 3.*P < 0.05, **P < 0.01, ***P < 0.001 compared with Control; # P < 0.05, ## P < 0.01, ### P < 0.001 compared with Model; & P < 0.05, && P < 0.01, &&& P < 0.001 compared with QS; ^P < 0.05, ^^P < 0.01 compared with Mcc950.

Article Snippet: The sections were then incubated overnight at 4°C with the following primary antibodies: NF-κB p65 Monoclonal antibody (66535-1-Ig, Proteintech, China), NLRP3 Monoclonal antibody (68102-1-Ig-100, Proteintech, China), Caspase-1 antibody (sc-56036, Santa Cruz, USA), and GSDMD Polyclonal antibody (20770-1-AP, Proteintech, China).

Techniques: Control

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Sustained activation of NF-κB through constitutively active IKKβ leads to senescence bypass in murine dermal fibroblasts.

doi: 10.1080/15384101.2024.2325802

Figure Lengend Snippet: Figure 2. Expression of constitutively active IKKβ bypasses cellular senescence. (a) Control (R26StopFLIKK2CA Cre-negative) and IKKβ-CA (R26StopFLIKK2CA Sm22αCre) skin fibroblasts were treated with MG132 (10 μM), a proteasome inhibitor, for 30 min prior to stimulation with IL-1β (10 ng/ml) for 5 min. Whole-cell lysates were analyzed by Western blotting using the indicated antibodies. Representative images are shown (n = 3). (b) Nuclear and cytoplasmic fractions were prepared from control and IKKβ-CA skin fibroblasts and analyzed by Western blotting. Nuclear p65 levels were quantified by ImageJ and normalized to the level of Lamin A (n = 3). (c)(d) Growth curves of control and IKKβ-CA skin fibroblasts under 20% and 3% oxygen conditions (n = 5). P2 Skin fibroblasts isolated from five adult control and IKKβ-CA mice were passaged in parallel under 20% oxygen conditions according to a 3T3 protocol and under 3% oxygen conditions according to a 3T1 protocol. The graph shows the cumulative number of cells in sequential passages. (e)(f) Representative images and quantification of senescence-associated β- galactosidase (SA-β-gal) staining of control and IKKβ-CA cells in passage 8 cultured under 3% and 20% oxygen conditions (n = 5). Scale bars, 100 µm. Error bars represent the standard error of the mean. (*p value < 0.05, **p value < 0.01)

Article Snippet: Phospho-p53 (Ser20) (# PA5–104741), phospho-p53 (Ser37) (#HYP80843) and phospho-RelA/p65 (Ser276) (#NB100–82086) antibodies were purchased from Invitrogen, MedChemExpress and Novus Biologicals, respectively.

Techniques: Expressing, Control, Western Blot, Isolation, Staining, Cell Culture

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Sustained activation of NF-κB through constitutively active IKKβ leads to senescence bypass in murine dermal fibroblasts.

doi: 10.1080/15384101.2024.2325802

Figure Lengend Snippet: Figure 3. Expression of nondegradable IκBα abolishes IKKβ-CA-induced senescence bypass. (a) Control (R26StopFLIKK2CA Cre-negative) and IKKβ-CA (R26StopFLIKK2CA Sm22αCre) skin fibroblasts were infected with lentivirus carrying an empty vector (EV) or FLAG-tagged IκBαSR. The expression of IκBαSR was confirmed by Western blotting using an antibody against the DYKDDDDK Tag. Representative images are shown (n = 4). (b) Nuclear and cytoplasmic fractions were prepared from control and IKKβ-CA skin fibroblasts harboring EV or IκBαSR. The nuclear p65 and p50 levels were measured by Western blotting, with quantification performed with ImageJ and normalized to the level of Lamin A (n = 4). (c)(d) Growth curves of control and IKKβ-CA skin fibroblasts harboring EV and IκBαSR cultured under 20% and 3% oxygen conditions, respectively. Skin fibroblasts were cultured under physiological oxygen level (3-5% oxygen) conditions until passage 3, at which time, they were infected with lentivirus. The cells were passaged again and treated with 2 μg/ml puromycin for 3 days under 3% oxygen conditions. The selected cells were passaged under 20% oxygen conditions according to a 3T2 protocol (c) or maintained under physiological oxygen level (3% oxygen) conditions according to a 3T1 protocol (d). The graph shows the cumulative number of cells in sequential passages. (e) Representative images and quantification of senescence-associated β-galactosidase (SA-β-gal) staining of control and IKKβ-CA cells harboring EV or IκBaSR cultured under 20% oxygen conditions. Scale bars, 100 µm. Error bars represent the standard error of the mean. (*p value < 0.05, **p value < 0.01)

Article Snippet: Phospho-p53 (Ser20) (# PA5–104741), phospho-p53 (Ser37) (#HYP80843) and phospho-RelA/p65 (Ser276) (#NB100–82086) antibodies were purchased from Invitrogen, MedChemExpress and Novus Biologicals, respectively.

Techniques: Expressing, Control, Infection, Plasmid Preparation, Western Blot, Cell Culture, Staining

Journal: Signal Transduction and Targeted Therapy

Article Title: Melatonin enhances radiofrequency-induced NK antitumor immunity, causing cancer metabolism reprogramming and inhibition of multiple pulmonary tumor development

doi: 10.1038/s41392-021-00745-7

Figure Lengend Snippet: Combined treatment with RFA and MLT reduced tumor malignancy in non-ablated areas, altered tumor metabolism, and the tumor microenvironment. a The integrative multi-omics analysis of cancer proliferation and malignancy-related pathways. Heatmap of alteration pathways in the transcriptomic and proteomic subtypes. Left, alteration pathways identified from the transcriptome. Right, alteration pathways are identified from the proteome. Color of each cell represents the average ssGSEA enrichment scores of that subtype; red denotes activation and blue denotes inhibition. Blank cells represent non-enrichment. b The validation of cancer proliferation and malignancy-related pathways. Western blot analysis of P53 (Cleaved-BID and P53), Wnt (C-myc, β-catenin, P-β-catenin, and Cyclin D1), Hedgehog (β-Arrestin 1 and Gli1), MAPK (P-erk, Foxo3, PKC, ERK2, p-STAT3 and STAT3), and NFкB (Rela, BTK, CSNK2A1) pathway-altered proteins in the RFA + MLT, RFA, MLT, and Control groups; The normalized altered gene expression of c P53 (Mdm4, Siva1, Fas, and Igfbp3), d NFкB (Ptgs2, Cxcl1, and Il1b), e Wnt (Sox17), and f MAPK (Hspa1b, Pgf, Fas, Angpt2, Cacna1g, Kit, Hspa1a, Map2k6, Dusp4, and Il1b) signal pathways in the RFA + MLT, RFA, MLT, and Control groups (*P < 0.05; **P < 0 .01; ***P < 0. 001)

Article Snippet: After that, the membranes were treated with monoclonal antibodies against Cleaved-BID (2003, CST), P53 (48818, CST), BTK (56044, CST), C-myc (18583, CST), β-catenin (8480, CST), P-β-catenin (4176, CST), Cyclin D1 (55506, CST), β-Arrestin 1 (12697, CST), Gli1 (2553, CST), P-erk (3371, CST), Foxo3 (2497, CST), PKCα (2056, CST), ERK2 (9108,CST), STAT3 (12640, CST), p-STAT3 (9145, CST) Rela (8214, CST), BTK (56044, CST), CSNK2A1 (2656, CST), and an internal control protein (β-actin) overnight at 4 °C (1:1000; Santa Cruz Biotechnology).

Techniques: Biomarker Discovery, Activation Assay, Inhibition, Western Blot, Control, Gene Expression

Journal: Oncogene

Article Title: Cyclin E and Bcl-xL cooperatively induce cell cycle progression in c-Rel-/- B cells.

doi: 10.1038/sj.onc.1206917

Figure Lengend Snippet: Figure 6 c-Rel regulates E2F3a expression in B cells. (a) Resting splenic B cells from c-rel þ / þ; bcl-xLTg (WT Tg) and c-rel/; bcl- xLTg (KO Tg) B cells were cultured for 36 h in the presence of 10 mg/ml anti-IgM and assayed by RT–PCR using primers specific for E2F3a. (b) Approximately 1 kb of mouse e2f3a promoter sequence is shown with two putative NF-kB/Rel-binding sites indicated by red boxes, as determined by Transfac database analysis. Site I corresponds to 864 bp upstream of the ATG translational start site ( þ 1) and site II is located 214 bp upstream. (c) Cells were cultured as described in (b) and assayed for NF-kB/Rel-binding activity by gel-shift assay using a radi- olabeled probe containing the 864 kB site and antibodies specific for p50, p65, and c-Rel to supershift complexes. The inducible complex (labeled A) is supershifted by p50, c-Rel, and p65 antibodies in wild-type B cells stimulated with 10 mg/ml anti-IgM, but a mutated site (864 kBMUT) prevents the binding of these complexes. (Supershifted complexes do not visibly migrate, but appear lighter than nonshifted control bands instead.) An additional site (214 kB) does not appear to bind any NF-kB/ Rel complexes either. Complex B represents nonspecific binding activity that serves to show equal loading of lanes. (d) CH31 B cells stimulated with 10 mg/ml anti-CD40 also exhibit c-Rel and p50 binding activity (labeled A) at the 864 kB site of the e2f3a promoter but a mutant 864 kB site and the 214 kB site do not bind. (e) WEHI 231 B cells stimulated with 10 mg/ml anti-CD40 show NF-kB/Rel binding activity (labeled A) while the mutant 864 kB site and 214 kB site do not exhibit any NF-kB/Rel- binding activity. (f) A total of 1 105 NIH 3T3 cells were transfected with 0.5 mg of firefly luciferase constructs containing a 2 kb e2f3a promoter (E2F3a-luc), a mutant 2 kb promoter (MUT- luc) bearing a mutation at the 864 kB site, or a positive control Igk chain promoter (Igk-luc), and cotransfected with either 0.5 mg of p50, c-Rel, or empty expression vector (pcDNA3). Cells were also cotransfected with a Renilla luciferase internal control plasmid containing a thymidine kinase promoter (tk-pRL) to drive constitutive Renilla luciferase activity for normalization of data. At 48 h, cells were harvested and assayed for firefly and Renilla luciferase activity. All experiments were carried out in triplicate and repeated in three separate experiments. A representative set of data is shown

Article Snippet: Supershift analysis was performed using 5–7mg anti-p130 (sc-317 ), anti-p107 (sc-317 ), anti-cRel (sc-071) Santa Cruz Biotechnology, anti-p50 from previous studies (Liou et al., 1994), and anti-p65 (RelA; 100–4165) from Rockland.

Techniques: Expressing, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Sequencing, Binding Assay, Activity Assay, Gel Shift, Labeling, Control, Mutagenesis, Transfection, Luciferase, Construct, Positive Control, Plasmid Preparation

Journal: International Journal of Molecular Sciences

Article Title: In Vitro and In Vivo Validation of Endothelium-Derived Potential Therapeutics for Myocardial Ischemia/Reperfusion Injury Identified by an AI-Enhanced Single-Cell and Virtual-Cell Paradigm

doi: 10.3390/ijms27062743

Figure Lengend Snippet: Andrographolide inhibits the S100A8 downstream IL-17 signaling pathway and reduces the inflammatory response of H/R-induced CMECs. ( A , B ) The expressions of IL-17A, IL-17RA, Act1, and NF-κB proteins were determined by Western blot ( n = 3). ( C – E ) The levels of IL-1β, IL-6, and TNF-α were determined through ELISA. ( F – H ) The expressions of IL-17A, IL-17RA and Act1 proteins were determined by Western blot in co-transfection with the overexpression S100A8 plasmid ( n = 3). Data are presented as mean ± SD ( n = 6). “Con or C” stands for the control group, “H/R” or “H” indicates the H/R group, and “H + AG or H + A” means the H/R group treated with AG. “H + A + S” means the H/R group treated with AG and overexpression of S100A8. ** p < 0.01 compared to the Con group; # p < 0.05, ## p < 0.01 compared to the H/R or H group, @ p < 0.05, @@ p < 0.01 compared to the H + A + S group.

Article Snippet: The membranes were blocked for 1 h at room temperature with 5% skimmed milk before being incubated overnight at 4 °C with the following primary antibodies: S100A8 (A15315, Abclonal, Wuhan, China), IL-17A (A12454, Abclonal, Wuhan, China), IL-17RA (A10052, Abclonal, Wuhan, China), Act1 (A6776, Abclonal, Wuhan, China), p-NF-κB p65 (1:500, AP0124, Abclonal, Wuhan, China), NF-κB p65 (1:500, A00284-1, Boster, Wuhan, China), β-actin (1:2000, TA-09, Zhongshan Golden Bridge, Beijing, China), and GAPDH (1:2000, TA-08, Zhongshan Golden Bridge, Beijing, China).

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Cotransfection, Over Expression, Plasmid Preparation, Control