Journal: Microbiology Spectrum

Article Title: Epstein-Barr Virus Envelope Glycoprotein gp110 Inhibits IKKi-Mediated Activation of NF-κB and Promotes the Degradation of β-Catenin

doi: 10.1128/spectrum.00326-23

Figure Lengend Snippet: gp110 disrupts IKKi-mediated activation of the NF-κB pathway. (A) HEK293T cells were transfected with gp110-HA expression plasmid or vector along with or without IKKi-Flag expression plasmid to activate the phosphorylation of p65 (Ser536). At 24 h posttransfection, the cell lysates were collected for WB with rabbit anti-p65 pAb, mouse anti-Flag MAb, and mouse anti-HA MAb. Rabbit anti-p65 (Ser536) pAb was used to detect the phosphorylation of p65, and β-actin was used as the loading control. (B and C) gp110-HA expression plasmid or vector was individually transfected into HEK293T cells (C) or cotransfected with the plasmid combination of IKKi-Myc/p65-Flag into HEK293T cells (B). At 24 h posttransfection, cells were infected with or without 100 HAU/mL SeV for 16 h, collected, and lysed. The samples were then used for Co-IP analysis with mouse anti-Myc MAb (B) or rabbit anti-p65 pAb (C). Immunoprecipitated proteins were then resolved by 10% SDS-PAGE, and WB was performed with mouse anti-Flag MAb, mouse anti-Myc MAb, and mouse anti-HA MAb. Rabbit anti-IKKi pAb and rabbit anti-p65 pAbs were used to detect the expression of endogenous IKKi and p65 (C), respectively, and β-actin was used as the loading control. (D) HEK293T cells were transfected with different concentrations of gp110-HA expression plasmid or vector. At 24 h posttransfection, cells were infected with or without 100 HAU/mL SeV for 16 h. Cell lysates were collected for WB with rabbit anti-p65 pAb and mouse anti-HA MAb. Rabbit anti-p65 (Ser536) pAb was used to detect the phosphorylation of p65, and β-actin was used as the loading control. (E) Hone1-EBV cells were transfected with the expression plasmid of shBALF4 or pSuper vector. At 24 h posttransfection, cells were treated with or without TPA (40 ng/mL) and NaB (3 mM) for 24 h to induce lytic EBV infection. Cells were then infected with or without 100 HAU/mL SeV for 16 h, and cell lysates were collected for WB with mouse anti-gp110 MAb, rabbit anti-p65 pAb, and rabbit anti-p65 (Ser536) pAb. β-Actin was used as the loading control. (F) HEK293T cells were transfected with gp110-HA expression plasmid or vector. At 24 h posttransfection, cells were infected with or without 100 HAU/mL SeV for 16 h. Cell lysates were harvested for cellular fractionation, and WB was performed with mouse anti-HA MAb and rabbit anti-p65 pAb. (G) Hone1-EBV cells were transfected with the expression plasmid of shBALF4 or pSuper vector. At 24 h posttransfection, cells were treated with or without TPA (40 ng/mL) and NaB (3 mM) for 24 h to induce lytic EBV infection. Cells were infected with or without 100 HAU/mL SeV for 16 h, and cell lysates were collected for WB with mouse anti-gp110 MAb and rabbit anti-p65 pAb. Here, GAPDH was used as the cytosol marker, and histone 3 was used as the nucleus marker. The gray analysis was calculated using ImageJ.

Article Snippet: Rabbit anti-ubiquitin MAb, anti-IRF3 (Ser396), and anti-p65 (Ser536) phosphorylated pAbs, alkaline phosphatase (AP)-labeled goat anti-mouse IgG, and goat anti-rabbit IgG were purchased from Cell Signaling Technology.

Techniques: Activation Assay, Transfection, Expressing, Plasmid Preparation, Phospho-proteomics, Control, Infection, Co-Immunoprecipitation Assay, Immunoprecipitation, SDS Page, Cell Fractionation, Marker

Journal:

Article Title: Influenza A Virus NS1 Protein Prevents Activation of NF-?B and Induction of Alpha/Beta Interferon

doi:

Figure Lengend Snippet: Activation of NF-κB by delNS1 virus infection in MEFs. (A) Detection of activated NF-κB by EMSA. MEFs were untreated (UT), mock infected, or infected with PR8, delNS1, or NDV viruses at an MOI of 1 for the indicated times. Nuclear extracts were subjected to EMSA with a DNA probe specific for NF-κB. TNF-α- and dsRNA-treated cells were included as positive controls. (B) Supershift of NF-κB complexes. Anti-p65 and anti-p50 antibodies (Ab) were used to shift the NF-κB complexes in delNS1- or NDV-infected MEFs at 6 h p.i.

Article Snippet: Cells were washed with PBS and fixed with ice-cold 100% methanol for 10 min. After blocking for 30 min with 3% bovine serum albumin (BSA) (Sigma), cells were incubated with rabbit anti-p65 antibody (Rockland) diluted 1:200 and mouse anti-NP antibody HT103 ( 45 ) diluted 1:500 in 3% BSA–PBS for 40 min. MEFs were washed three times with PBS before incubation with fluorescein isothiocyanate-conjugated antirabbit antibody and Texas red-conjugated antimouse antibody (Boehringer Mannheim) diluted 1:500 in 3% BSA–PBS.

Techniques: Activation Assay, Infection

Journal: British Journal of Nutrition

Article Title: Cocoa polyphenols prevent inflammation in the colon of azoxymethane-treated rats and in TNF-α-stimulated Caco-2 cells

doi: 10.1017/s0007114512004862

Figure Lengend Snippet: Fig. 2. Effect of the cocoa-enriched diet on the colonic expression of cyclo-oxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS) and NF-kB induced by azoxymethane (AOM). (A) Representative RT-PCR analysis and percentage values of mRNA levels of COX-2 and iNOS in the distal colon mucosa of rats injected with saline or AOM and fed with the control or cocoa-enriched diet. (B) Representative photographs for immunohistochemical staining of NF-kB subunit p65 (dark brown)-positive cells (400£ magnification) and the percentage score in colon tissues from rats injected with saline or AOM and fed the control or cocoa-enriched diet. Values are means from six to eight rats in each group, with standard deviations represented by vertical bars. a,b,c Mean values with unlike letters were signifi- cantly different (P,0·05). GADPH, glyceraldehyde 3-phosphate. , Control; , control þ AOM; , cocoa; , cocoa þ AOM.

Article Snippet: Anti-p38, anti-p65 (NF-kB), anti-iNOS and anti-COX-2 were purchased from Santa Cruz Biotechnology.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Injection, Saline, Control, Immunohistochemical staining, Staining

Journal: British Journal of Nutrition

Article Title: Cocoa polyphenols prevent inflammation in the colon of azoxymethane-treated rats and in TNF-α-stimulated Caco-2 cells

doi: 10.1017/s0007114512004862

Figure Lengend Snippet: Fig. 5. Effect of cocoa polyphenolic extract (CPE) on NF-kB translocation and phosphorylated levels of extracellular signal-regulated kinases (ERK), c-Jun NH2- terminal kinases (JNK) and p38 MAPK (p38) induced by TNF-a in Caco-2 cells. C ( ): control, untreated cells; TNF-a ( ): cells treated with 40 ng/ml TNF-a for 1 h; CPE ( ): cells treated with 10 mg/ml CPE for 20 h; CPE þ TNF-a ( ): cells pretreated with 10 mg/ml CPE for 20 h and then treated with 40 ng/ml TNF-a for 1 h. NF-kB levels were determined by Western blot in the nuclear or cytosolic cellular compartment. Values are means, with standard deviations represented by vertical bars. (A) Representative bands of three different experiments. (B) Percentage values of nuclear ( ) and cytosolic NF-kB ( ) levels relative to the control condition. Phosphorylated and total levels of ERK, JNK and p38 were determined by Western blot analysis using phospho- and total specific antibodies. (C) Bands are representative of two to three different experiments. (D) Percentage values of the p-ERK:ERK, p-JNK:JNK and p-p38:p38 ratios relative to the control condition. a,b,c,d Mean values with unlike letters were significantly different (P,0·05).

Article Snippet: Anti-p38, anti-p65 (NF-kB), anti-iNOS and anti-COX-2 were purchased from Santa Cruz Biotechnology.

Techniques: Translocation Assay, Control, Western Blot