Journal: BioMed Research International

Article Title: Galangin Suppresses Renal Inflammation via the Inhibition of NF- κ B, PI3K/AKT and NLRP3 in Uric Acid Treated NRK-52E Tubular Epithelial Cells

doi: 10.1155/2019/3018357

Figure Lengend Snippet: Effect of galangin on the expression of the phosphorylated form of PI3K and AKT in UA treated NRK-52E cells. Galangin decreased the expression of the phosphorylated form of PI3K and AKT in UA treated NRK-52E cells. Each of the protein bands was normalized to band of β -actin and reported as relative intensity. Results are expressed as mean ± SEM of three independent experiments. #p < 0.05, ##p < 0.01 vs control group; ∗ p < 0.05, ∗∗ p < 0.01 vs UA group.

Article Snippet: The antibodies used were the following: rabbit monoclonal anti-p-I κ B α (#2859S), rabbit polyclonal anti-I κ B α (#9242S), rabbit polyclonal anti-p-p65(#3031S), rabbit monoclonal anti-p65(#8242S), rabbit monoclonal anti-p-Ikk β (#2078S), rabbit monoclonal anti-Ikk β (#8943S), rabbit monoclonal anti-NLRP3(#15101S), rabbit monoclonal anti-ASC(#67824S), rabbit polyclonal anti-caspase-1(#2225S), rabbit polyclonal anti-p-PI3K(#4228S), rabbit polyclonal anti-PI3K(#4292S), mouse monoclonal anti-p-AKT(#4051S), mouse monoclonal anti-AKT(#2920S), and rabbit polyclonal anti- β -actin(#4967S), and purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Expressing

Journal: PLoS ONE

Article Title: How the Venom from the Ectoparasitoid Wasp Nasonia vitripennis Exhibits Anti-Inflammatory Properties on Mammalian Cell Lines

doi: 10.1371/journal.pone.0096825

Figure Lengend Snippet: L929sA cells stably transfected with a NF-κB-dependent reporter gene were pretreated with indicated concentrations of venom for 15 minutes followed by stimulation for 6 hours with TNF (2000 IU/ml). Luciferase activity was normalized for β-galactosidase expression. The data are expressed as the mean ±SDs of three biological replicates. Normality was confirmed by a Shapiro-Wilk test (W = 0.9689). * p<0.05, ** p<0.01 versus TNF alone, ANOVA with Bonferroni posthoc test.

Article Snippet: Antibodies to rabbit phospho-IKKα (ser180)/(ser181)β, rabbit phospho-NF-κB p65(ser536), rabbit phospho-ERK MAPK and rabbit phospho-p38 MAPK were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Stable Transfection, Transfection, Luciferase, Activity Assay, Expressing

Journal: PLoS ONE

Article Title: How the Venom from the Ectoparasitoid Wasp Nasonia vitripennis Exhibits Anti-Inflammatory Properties on Mammalian Cell Lines

doi: 10.1371/journal.pone.0096825

Figure Lengend Snippet: A. Western blot analysis of nuclear extracts from Raw264.7. After pre-incubation with N. vitripennis venom (5 µg/ml) for 15 minutes, cells were induced for the indicated times with LPS (1 µg/ml). Subsequently, nuclear extracts were subjected to Western blot analysis to determine p65 levels. Separation of nuclear and cytoplasmic fractions was verified using PARP as control for the nuclear fractions. B. After 15 minutes pre-incubation with N. vitripennis venom, cells were induced for the indicated times with LPS (1 µg/ml). Immunofluorescence staining was performed to visualize the trafficking of the p65 subunit. Representative results from three separate experiments are shown.

Article Snippet: Antibodies to rabbit phospho-IKKα (ser180)/(ser181)β, rabbit phospho-NF-κB p65(ser536), rabbit phospho-ERK MAPK and rabbit phospho-p38 MAPK were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Western Blot, Incubation, Immunofluorescence, Staining

Journal: PLoS ONE

Article Title: How the Venom from the Ectoparasitoid Wasp Nasonia vitripennis Exhibits Anti-Inflammatory Properties on Mammalian Cell Lines

doi: 10.1371/journal.pone.0096825

Figure Lengend Snippet: HEK293T cells were transiently transfected by the PEI method with 100(Gal)2-50.huIL6P-luc+ and with various expression plasmids, the total amount of DNA being fixed at 250 ng, i.e., pGal4 (10 ng) or pGal4-p65 (10 ng) whether or not with pGR (25 ng). 10 −6 M DEX or 5 µg/ml of venom was added 24 hours before analysis. Lysates were made and the relative luciferase activity was determined by using β-gal values as a basis for normalization. The data are expressed as the mean ±S.D. of three biological replicates.

Article Snippet: Antibodies to rabbit phospho-IKKα (ser180)/(ser181)β, rabbit phospho-NF-κB p65(ser536), rabbit phospho-ERK MAPK and rabbit phospho-p38 MAPK were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Transfection, Expressing, Luciferase, Activity Assay

Journal: PLoS ONE

Article Title: How the Venom from the Ectoparasitoid Wasp Nasonia vitripennis Exhibits Anti-Inflammatory Properties on Mammalian Cell Lines

doi: 10.1371/journal.pone.0096825

Figure Lengend Snippet: TNF stimulation leads to activation of the canonical NF-κB signal transduction pathway marked by IκBα degradation and translocation of the p65-p50 dimer to the nucleus. Subsequent NF-κB DNA binding enables transcription of different genes, among which the cytokine IL-6. Venom treatment leads to the inhibition of TNF induced IL-6 gene expression. In addition, TNF activates the MAPKs,JNK, ERK1/2 and p38, and pretreatment with venom results in prolonged JNK activation. The negative NF-κB regulators A20 and IκBα are indicated and both were suppressed by the venom. Glucocorticoid binding to the cytosolic Glucocorticoid Receptor (GR) results in the dissociation of chaperoning proteins, followed by GR translocation to the nucleus, where it can interfere with the activity of NF-κB. Activated GR can also directly bind to the DNA, stimulating transcription of FKBP5, MKP1 and GILZ target genes. Only the two latter genes are transcriptionally induced by the venom. The red arrows mark the various levels at which venom of N. vitripennis interferes with the represented cellular signaling pathways.

Article Snippet: Antibodies to rabbit phospho-IKKα (ser180)/(ser181)β, rabbit phospho-NF-κB p65(ser536), rabbit phospho-ERK MAPK and rabbit phospho-p38 MAPK were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Activation Assay, Transduction, Translocation Assay, Binding Assay, Inhibition, Expressing, Activity Assay

Journal: Brain Sciences

Article Title: Electroacupuncture Alleviates Neuroinflammation by Inhibiting the HMGB1 Signaling Pathway in Rats with Sepsis-Associated Encephalopathy

doi: 10.3390/brainsci12121732

Figure Lengend Snippet: EA modulated HMGB1 signaling in the hippocampus: n = 5 per group. ( A ) Representative blots of HMGB1. ( B ) Representative blots of TLR4. ( C ) Representative blots of RAGE. ( D ) Representative blots of p-NF-κB p65 and NF-κB p65. ( E ) Quantification of the expression level of HMGB1. ( F ) Quantification of the expression level of TLR4. ( G ) Quantification of the expression level of RAGE. ( H ) Quantification of the ratio of p-NF-κB p65 and NF-κB p65. Data are presented as means ± SEM. Compared with the sham group, ** p < 0.01, *** p < 0.001, and **** p < 0.0001; compared with the LPS group, ## p < 0.01, ### p < 0.001, and #### p < 0.0001. ■, ▲, ▼, ◆: Represents the individual value of the rats in each group. Abbreviations: HMGB1, high mobility group box 1 protein; NF-κB, nuclear factor-κB; p- NF-κB, phosphorylated-NF-κB; RAGE, the receptor for advanced glycation end products; TLR4, Toll-like receptor 4; EA, electroacupuncture; LPS: lipopolysaccharide; ns: no significant difference.

Article Snippet: After blocking with 5% skim milk or 5% BSA in Tris-buffered saline and Tween 20 (TBST, 0.1%) at room temperature, the membranes were incubated at 4 °C overnight with primary antibodies: rabbit anti-HMGB1 antibody (A2553, 1:1000, Abclonal, Wuhan, China), rabbit anti-glial fibrillary acidic protein (GFAP) antibody (A0237, 1:1000, Abclonal, Wuhan, China), rabbit anti-Occludin antibody (A2601, 1:1000, Abclonal, Wuhan, China), rabbit anti-connexin 43 (Cx43) antibody (A11752, 1:2000, Abclonal, Wuhan, China), rabbit anti-TNF-α antibody (A0277, 1:1500, Abclonal, Wuhan, China), rabbit anti-ionized calcium-binding adapter molecule 1 (Iba1) antibody (A19776, 1:500, Abclonal, Wuhan, China), rabbit anti-zonula occludens-1 (ZO-1) antibody (AF5145, 1:1000, Affinity Biosciences, OH, USA), rabbit anti-TLR4 antibody (AF7017, 1:2000, Affinity Biosciences, OH, USA), rabbit anti-IL-1β antibody (A20529, 1:1000, Abclonal, Wuhan, China), rabbit anti-NF-κB antibody (A19653, 1:2000, Abclonal, Wuhan, China), rabbit anti-RAGE antibody (A13264, 1:1500, Abclonal, Wuhan, China), rabbit anti-Phospho-NF-κB p65 (Ser536) antibody (3033, 1:1000, Cell Signaling Technology, Boston, MA, USA), rabbit anti-IL-6 antibody (21865-1-AP, 1:1000, Proteintech, Wuhan, China), mouse anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody (10494-1-AP, 1:8000, Proteintech, Wuhan, China), and mouse anti-β-Actin monoclonal antibody (66009-1-lg, 1;8000, Proteintech, Wuhan, China).

Techniques: Expressing

Journal: International Journal of Molecular Sciences

Article Title: Effect of LDHA Inhibition on TNF-α-Induced Cell Migration in Esophageal Cancers

doi: 10.3390/ijms232416062

Figure Lengend Snippet: The effect of sodium oxamate on activation of TNF-α-induced MMP9-related ERK1/2 and NFκB signaling pathways in esophageal cancer cells. SO-dependent downregulation of MMP9 in response to TNF-α, seen in gelatin zymography (upper), and time corse of ERK1/2 and NFκB/p65 phosphorylation after TNF-α stimulation in ( A ) KYSE150 cells and ( B ) EC7 cells treated or not with SO (50 mM) for 24 h, visualized by Western blotting. Quantitative analysis using the differential densitometry of TNF-α-induced ERK1/2(Thr202/Tyr204) and NFκB/p65(Ser536) activation in the form of bar graphs, showing the ratio of phospho-proteins normalized to total protein in SO-treated and non-treated cells (right panels). Ctrl—control; SO—sodium oxamate; ACTB—β-actin/loading control; * p < 0.001.

Article Snippet: Rabbit anti-β-Actin (4970), rabbit anti-HIF-1α (36169), mouse anti-ERK1/2 (4696), rabbit anti-Phospho-ERK1/2 (Thr202/Tyr204) (4370), mouse anti-NFκB p65 (6956), rabbit anti-Phospho-NFκB p65 (Ser536) (3033), rabbit anti-Phospho-LDHA (Tyr10) (8176) antibodies were products of Cell Signaling.

Techniques: Activation Assay, Zymography, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Effect of LDHA Inhibition on TNF-α-Induced Cell Migration in Esophageal Cancers

doi: 10.3390/ijms232416062

Figure Lengend Snippet: The effect of LDHA gene silencing on activation of TNF-α-induced MMP9-related ERK1/2 and NFκB signaling pathways in esophageal cancer cells. LDHA siRNA-associated downregulation of MMP9 in response to TNF-α in KYSE150 cells, seen in gelatin zymography (upper) and levels of total and phosphorylated ERK1/2 and NFκB/p65 proteins in ( A ) KYSE150 cells ( B ) and EC7 cells transfected with LDHA siRNA, subjected or not to hourly TNF-α stimulation (30 ng/mL), visualized by Western blotting. Quantitative analysis using the differential densitometry of TNF-α-induced ERK1/2 activation in the form of bar graphs, showing the ratio of phospho-ERK1/2 (Thr202/Tyr204) normalized to total ERK1/2 in LDHA siRNA-transfected and control cells (bottom panels). Ctrl—control; si_LDHA—LDHA siRNA; sc—scrambled sequence siRNA; * p < 0.05; ** p < 0.005.

Article Snippet: Rabbit anti-β-Actin (4970), rabbit anti-HIF-1α (36169), mouse anti-ERK1/2 (4696), rabbit anti-Phospho-ERK1/2 (Thr202/Tyr204) (4370), mouse anti-NFκB p65 (6956), rabbit anti-Phospho-NFκB p65 (Ser536) (3033), rabbit anti-Phospho-LDHA (Tyr10) (8176) antibodies were products of Cell Signaling.

Techniques: Activation Assay, Zymography, Transfection, Western Blot, Sequencing

Journal: BioMed Research International

Article Title: Hyperbranched Cationic Glycogen Derivative-Mediated I κ B α Gene Silencing Regulates the Uveoscleral Outflow Pathway in Rats

doi: 10.1155/2020/8206849

Figure Lengend Snippet: Nucleotide sequences of primers for PCR.

Article Snippet: The former were used for immunofluorescence staining of NF- κ Bp65, as described above, at an anti-NF- κ Bp65 rabbit monoclonal antibody dilution of 1 : 50 (Cell Signaling Technology, Danvers, USA).

Techniques: Sequencing

Journal: BioMed Research International

Article Title: Hyperbranched Cationic Glycogen Derivative-Mediated I κ B α Gene Silencing Regulates the Uveoscleral Outflow Pathway in Rats

doi: 10.1155/2020/8206849

Figure Lengend Snippet: Examination of NF- κ Bp65 mRNA and protein levels at 24, 48, and 72 h after RNAi. The error bars represent the standard deviation calculated from three parallel experiments ( n = 6 per group). Notes . (a) NF- κ Bp65 mRNA expression was quantified by real-time PCR. Expression levels were normalized with GAPDH. (b) NF- κ Bp65 protein expression was examined by western blot. The NF- κ Bp65 and β -actin bands were analyzed by densitometry, and the values were normalized with β -actin, which were represented in the bar graph. Abbreviations: PBS—phosphate-buffered saline; DMAPA-Glyp—3-(dimethylamino)-1-propylamine-conjugated glycogen; NC—nonspecific control; Lipo—Lipofectamine™ 2000.

Article Snippet: The former were used for immunofluorescence staining of NF- κ Bp65, as described above, at an anti-NF- κ Bp65 rabbit monoclonal antibody dilution of 1 : 50 (Cell Signaling Technology, Danvers, USA).

Techniques: Standard Deviation, Expressing, Real-time Polymerase Chain Reaction, Western Blot

Journal: BioMed Research International

Article Title: Hyperbranched Cationic Glycogen Derivative-Mediated I κ B α Gene Silencing Regulates the Uveoscleral Outflow Pathway in Rats

doi: 10.1155/2020/8206849

Figure Lengend Snippet: NF- κ Bp65 nuclear translocation at 24, 48, and 72 h after RNAi by immunofluorescence (×400) ( n = 4 per group). Notes . (a) The white arrows indicate increased nuclear expression of NF- κ Bp65 in the DMAPA-Glyp+siRNA group; the asterisks indicate the weak nuclear signal of NF- κ Bp65 in the DMAPA-Glyp+NC group; the solid line rectangle indicates the magnifying region of the nuclear expression of NF- κ Bp65. (b) The fluorescence level counting of NF- κ Bp65 in the nuclei of the ciliary muscle was quantified to confirm NF- κ Bp65 nuclear translocation, and the values were represented in the bar graph. The error bars represent the standard deviation calculated from three parallel experiments. ∗ P < 0.01, compared with the DMAPA-Glyp+NC group. Abbreviations: DAPI—4,6-diamidino-2-phenylindole; cb—ciliary body; cm—ciliary muscle; scle—sclera; DMAPA-Glyp—3-(dimethylamino)-1-propylamine-conjugated glycogen; NC—nonspecific control.

Article Snippet: The former were used for immunofluorescence staining of NF- κ Bp65, as described above, at an anti-NF- κ Bp65 rabbit monoclonal antibody dilution of 1 : 50 (Cell Signaling Technology, Danvers, USA).

Techniques: Translocation Assay, Immunofluorescence, Expressing, Fluorescence, Standard Deviation

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Regulation of Cr(VI)-Induced Premature Senescence in L02 Hepatocytes by ROS-Ca 2+ -NF- κ B Signaling

doi: 10.1155/2022/7295224

Figure Lengend Snippet: Cr(VI) caused intracellular Ca 2+ overload and NF- κ B activation. The L02 hepatocytes were exposed to Cr(VI) for 4 weeks. (a) The mean fluorescence intensity intracellular Ca 2+ concentration was determined by light microscopy (the micrograph of the cells, 200x), and ImageJ software was used to analyze the relative fluorescent intensity showed in bar graph; (b) spectrofluorometry was used to detect the change of Ca 2+ concentration in cytoplasm. (c) Rhod-2 was used to assay the concentration of Ca 2+ in mitochondria. (d) The protein levels of p65 (cytoplasm and nucleus) were determined by Western blot. (e) The expression of I κ B α and IKK α was detected by Western blot after Cr(VI) treatment. ImageJ software was used to analyze the relative levels of proteins normalized to the expression of GAPDH. All experiments were repeated at least 3 times and expressed as mean ± SD. ∗ p < 0.05, compared with control. For the sake of clarity, the same control GAPDH was applied to compare with all experimentally relevant proteins with the same exposure time detected on the same SDS-PAGE gel unless otherwise stated.

Article Snippet: Antibodies against p21Waf1/Cip1 (#2947), PCNA (#13110), and Phospho-NF- κ B p65 (Ser536) (#3033) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Activation Assay, Fluorescence, Concentration Assay, Light Microscopy, Software, Western Blot, Expressing, SDS Page

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Regulation of Cr(VI)-Induced Premature Senescence in L02 Hepatocytes by ROS-Ca 2+ -NF- κ B Signaling

doi: 10.1155/2022/7295224

Figure Lengend Snippet: The intracellular Ca 2+ overload and NF- κ B activation caused by Cr(VI) depended on ROS accumulation. The L02 cells were pretreated with NAC for 1 h prior to Cr(VI) treatment for 4 weeks. (a) The intracellular Ca 2+ concentration was assayed by flow cytometry (histogram) and spectrofluorometry (bar graph). (b) The fluorescence of mitochondria Ca 2+ was quantitated via spectrofluorometry. (c) The protein levels of p65 (cytoplasm and nucleus) and (d) I κ B α and IKK α were determined by Western blot. ImageJ software was used to analyze the relative levels of proteins normalized to the expression of GAPDH. All experiments were repeated at least 3 times and expressed as mean ± SD. ∗ p < 0.05, compared with control; # p < 0.05, compared with Cr(VI)-exposed group. For the sake of clarity, the same control GAPDH was applied to compare with all experimentally relevant proteins with the same exposure time detected on the same SDS-PAGE gel unless otherwise stated.

Article Snippet: Antibodies against p21Waf1/Cip1 (#2947), PCNA (#13110), and Phospho-NF- κ B p65 (Ser536) (#3033) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Activation Assay, Concentration Assay, Flow Cytometry, Fluorescence, Western Blot, Software, Expressing, SDS Page

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Regulation of Cr(VI)-Induced Premature Senescence in L02 Hepatocytes by ROS-Ca 2+ -NF- κ B Signaling

doi: 10.1155/2022/7295224

Figure Lengend Snippet: Ca 2+ overload related to NF- κ B activation and proinflammatory cytokines secretion induced by Cr(VI). The L02 cells were pretreated with BAPTA-AM prior to Cr(VI) treatment for 4 weeks. (a) The cytoplasmic and mitochondrial Ca 2+ concentration was determined by spectrofluorometry. (b) β -Galactosidase Staining Kit (200x) (the percentage of senescent cells was showed in bar graph) and (c) DAPI staining were used to detect SAHF (200x). (d) The secretion of IL-6 and FGF23 were assayed by ELISA kit. (e) The protein levels of p65 (cytoplasm and nucleus) and (f) I κ B α and IKK α were determined by Western blot. ImageJ software was used to analyze the relative levels of proteins normalized to the expression of GAPDH. All experiments were repeated at least 3 times and expressed as mean ± SD. ∗ p < 0.05, compared with control; # p < 0.05, compared with Cr(VI)-exposed group. For the sake of clarity, the same control GAPDH was applied to compare with all experimentally relevant proteins with the same exposure time detected on the same SDS-PAGE gel unless otherwise stated.

Article Snippet: Antibodies against p21Waf1/Cip1 (#2947), PCNA (#13110), and Phospho-NF- κ B p65 (Ser536) (#3033) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Activation Assay, Concentration Assay, Staining, Enzyme-linked Immunosorbent Assay, Western Blot, Software, Expressing, SDS Page

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Regulation of Cr(VI)-Induced Premature Senescence in L02 Hepatocytes by ROS-Ca 2+ -NF- κ B Signaling

doi: 10.1155/2022/7295224

Figure Lengend Snippet: Active NF- κ B potentiates premature senescence without influencing intracellular Ca 2+ concentration. PDTC was used to pretreat the L02 hepatocytes before Cr(VI) exposure for 4 weeks. (a) p65 protein (cytoplasm and nucleus) levels were detected by Western blot, and ImageJ software was used to analyze the relative levels of proteins normalized to the expression of GAPDH. (b) β -Galactosidase Staining Kit (200x) and (c) DAPI staining were used to detect SAHF (200x). (d) IL-6 and FGF23 secretion was analyzed by ELISA kit. (e) The fluorescence of intracellular and mitochondria Ca 2+ was quantitated via spectrofluorometry. All experiments were repeated at least 3 times and expressed as mean ± SD. ∗ p < 0.05, compared with control; # p < 0.05, compared with Cr(VI)-exposed group.

Article Snippet: Antibodies against p21Waf1/Cip1 (#2947), PCNA (#13110), and Phospho-NF- κ B p65 (Ser536) (#3033) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Concentration Assay, Western Blot, Software, Expressing, Staining, Enzyme-linked Immunosorbent Assay, Fluorescence