Journal: Journal of Biological Chemistry

Article Title: C-type Lectin Receptor Dectin-3 Mediates Trehalose 6,6′-Dimycolate (TDM)-induced Mincle Expression through CARD9/Bcl10/MALT1-dependent Nuclear Factor (NF)-κB Activation

doi: 10.1074/jbc.m114.588574

Figure Lengend Snippet: FIGURE 2. Dectin-3 is required for TDM-induced NF-B activation. A–C, BMDMs from WT and Dectin-3 KO mice were either untreated or stimulated with plate-coated TDM for indicated time points. A, total cell lysates were prepared from these cells and then subjected to immunoblotting analysis using indicated antibodies. B, nuclear extracts were prepared and analyzed by immunoblotting with p65, NFAT-c1, and proliferating cell nuclear antigen antibodies. C, immunoblotting with the indicated antibodies were done with the total cell lysates. D, BMDMs from WT mice were pretreated with Dectin-2 or Dectin-3 blockingantibodiesfor45min.Resultingcellswerestimulatedwithplate-coated-mannan(man)orTDMfor1h.Nuclearextractswerepreparedandanalyzed by immunoblotting with p65 and proliferating cell nuclear antigen (PCNA) antibodies. Total cell lysates were immunoblotting with Dectin-2 or Dectin-3 antibodies. E, ELISA results for TNF-, IL-6, and IL-1 in supernatants of BMDMs derived from WT or Dectin-3 KO mice, which were stimulated with plate-coated TDM for 8 h. Data shown are representative of three independent experiments. S.D. is indicated. **, p 0.01 (t test). Ctr, control.

Article Snippet: Antibodies and Reagents—Antibodies against phospho-p38 (4631), phospho-ERK (9101), phospho-JNK (9251), phosphoIKK (2697), total p38 (9212), and total JNK (9252) were purchased from Cell Signaling Technology; antibodies against p65 (sc-8008), proliferating cell nuclear antigen (sc-56), NFAT-c1 (sc-7294), ERK (sc-154), IKK (sc-7218), I B (sc-371), FLAG (sc-807), and tubulin (sc-8035) were from Santa Cruz Biotechnology.

Techniques: Activation Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Derivative Assay, Control

Journal: Journal of Biological Chemistry

Article Title: C-type Lectin Receptor Dectin-3 Mediates Trehalose 6,6′-Dimycolate (TDM)-induced Mincle Expression through CARD9/Bcl10/MALT1-dependent Nuclear Factor (NF)-κB Activation

doi: 10.1074/jbc.m114.588574

Figure Lengend Snippet: FIGURE 4. NF-B p65 subunit binds with Mincle promoter to regulate Mincle expression. A, the structure for Mincle gene promoter region. 1589, 941, and 851 are the three predicted NF-B-binding sites. Different luciferase reporter constructs are also shown. B, BMDMs were stimulated with plate-coated TDM for 2 h or left untreated (0h), followed by ChIP assay with the indicated antibodies and real-time PCR analysis for Mincle promoter. Results are presented as means plus S.D. after normalization to input. C, RAW264.7 cells were transfected with different luciferase reporter constructs together with Dectin-3 expression plasmid. The luciferase activities of the Mincle promoter were measured. Two putative NF-B-binding sites were mutated and the mutant reporter was designated as 941m and 851m. Data shown are representative of two independent experiments. S.D. is indicated. **, p 0.01 (t test). Ctr, control.

Article Snippet: Antibodies and Reagents—Antibodies against phospho-p38 (4631), phospho-ERK (9101), phospho-JNK (9251), phosphoIKK (2697), total p38 (9212), and total JNK (9252) were purchased from Cell Signaling Technology; antibodies against p65 (sc-8008), proliferating cell nuclear antigen (sc-56), NFAT-c1 (sc-7294), ERK (sc-154), IKK (sc-7218), I B (sc-371), FLAG (sc-807), and tubulin (sc-8035) were from Santa Cruz Biotechnology.

Techniques: Expressing, Binding Assay, Luciferase, Construct, Real-time Polymerase Chain Reaction, Transfection, Plasmid Preparation, Mutagenesis, Control

Journal: Kidney Research and Clinical Practice

Article Title: Maternal exposure to airborne particulate matter during pregnancy and lactation induces kidney injury in rat dams and their male offspring: the role of vitamin D in pregnancy and beyond

doi: 10.23876/j.krcp.23.106

Figure Lengend Snippet: (A–D) Nuclear factor erythroid 2-related factor 2 (Nrf2), (E–H) nuclear factor-kappa B (NF-κB) p50, and (I–L) tumor necrosis factor alpha (TNF-α). Western blot analysis revealed decreases in Nrf2 (A) and NF-κB p50 (E) and an increase in TNF-α (I) in PM 2.5 -exposed dam kidneys as compared to the controls. Vitamin D (Vit D) supplementation reduced intrarenal TNF-α activity (I). While Nrf2 and NF-κB p50 expressions were well observed in control dam kidneys (B and F; arrows, respectively), they were weakly detected in PM 2.5 -exposed kidneys (C and G, respectively). Vit D intake did not restore their expressions (D and H). Intrarenal TNF-α expression was clearly detected throughout all tubular segments and glomeruli in the PM 2.5 group (K, arrows) as compared to the control group (J) and the PM 2.5 with Vit D group (L). (B–D, F–H, and J–L) Immunohistochemistry; ×200, bar = 50 µm (n = 3 for each group). * p < 0.05, control vs. PM 2.5 ; * * p < 0.05, control vs. PM 2.5 + Vit D; # p < 0.05, PM 2.5 vs. PM 2.5 + Vit D. PM, particulate matter.

Article Snippet: Primary antibodies against VDR (Santa Cruz Biotechnology), klotho (Invitrogen), CYP24A1 (Invitrogen), renin (Santa Cruz Biotechnology), ACE (Invitrogen), Nrf2 (Novus), NF-κB p50 (Santa Cruz Biotechnology), and TNF-α (Invitrogen) were used.

Techniques: Western Blot, Activity Assay, Control, Expressing, Immunohistochemistry

Journal: Kidney Research and Clinical Practice

Article Title: Maternal exposure to airborne particulate matter during pregnancy and lactation induces kidney injury in rat dams and their male offspring: the role of vitamin D in pregnancy and beyond

doi: 10.23876/j.krcp.23.106

Figure Lengend Snippet: (A–D) Nuclear factor erythroid 2-related factor 2 (Nrf2), (E–H) nuclear factor-kappa B (NF-κB) p50, (I) tumor necrosis factor alpha (TNF-α). Western blot analysis revealed decreases in Nrf2 (A) and NF-κB p50 (E) activities in pups from PM 2.5 -exposed dams as compared to the controls. Maternal vitamin D (Vit D) supplementation restored intrarenal NF-κB p50 activity in pup kidneys (E). While the Nrf2 and NF-κB p50 expressions were well observed in control pup kidneys (B and F; arrows, respectively), they were weakly detected in pup kidneys of maternal PM 2.5 exposure (C and G, respectively). Maternal Vit D intake did not restore Nrf2 expression in pups (D) whereas it increased the intrarenal expression of NF-κB p50 (H, arrows). (B–D and F–H) Immunohistochemistry; ×200, bar = 50 µm (n = 5 for each group). No difference was observed in the intrarenal activity of TNF-α (I) among the groups ( * p < 0.05, control vs. PM 2.5 ; # p < 0.05, PM 2.5 vs. PM 2.5 + Vit D). PM, particulate matter.

Article Snippet: Primary antibodies against VDR (Santa Cruz Biotechnology), klotho (Invitrogen), CYP24A1 (Invitrogen), renin (Santa Cruz Biotechnology), ACE (Invitrogen), Nrf2 (Novus), NF-κB p50 (Santa Cruz Biotechnology), and TNF-α (Invitrogen) were used.

Techniques: Western Blot, Activity Assay, Control, Expressing, Immunohistochemistry

Journal: Journal of Animal Science and Biotechnology

Article Title: Sulforaphane prevents LPS-induced inflammation by regulating the Nrf2-mediated autophagy pathway in goat mammary epithelial cells and a mouse model of mastitis

doi: 10.1186/s40104-023-00858-9

Figure Lengend Snippet: SFN inhibited expression of inflammatory factors and mediators and NF-κB (p65) activation in LPS-induced primary GMECs. Cells were pretreated with different concentrations of SFN (1.25, 2.5 and 5 μmol/L) for 12 h in the absence or presence of LPS (10 μg/mL) for 6 h. A GMECs were treated SFN for 24 h and the cytotoxicity of SFN was detected using CCK-8 assay ( n = 4). B–D qRT-PCR analysis of relative mRNA levels of inflammatory factors ( n = 3). E–G Western blot analysis of COX2 and iNOS levels ( n = 3). H–J Western blot analysis of p-IκBα, IκBα, p-p65 and p65 ( n = 3). K Immunofluorescent analysis of NF-κBp65 and nuclear staining with DAPI (blue) in GMECs pretreated with SFN (5 μmol/L) for 12 h in the absence or presence of LPS (10 μg/mL) for 6 h (Bar = 10 μm). One-way ANOVA, Dunnett's post-hoc test. Data are presented as the mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001

Article Snippet: The primary antibodies used in this study included anti-NFE2L2/Nrf2 (1:400, ab137550, Abcam, Cambridge, USA), anti-NF-κB p65 (1:200, 10745–1-AP, Proteintech, Wuhan, China) and LC3 (1:300, 14600–1-AP, Proteintech).

Techniques: Expressing, Activation Assay, CCK-8 Assay, Quantitative RT-PCR, Western Blot, Staining

Journal: Cell reports

Article Title: IFIT1 Exerts Opposing Regulatory Effects on the Inflammatory and Interferon Gene Programs in LPS-Activated Human Macrophages

doi: 10.1016/j.celrep.2018.09.002

Figure Lengend Snippet: (A) Control and IFIT1 shRNA-expressing THP1 cells were stimulated with 100 ng/mL LPS for the indicated times, and cytosolic and nuclear fractions were subjected to western blot analysis for phos-pho-p65/NF-κB (Ser536), phospho p38 and MAPK (Thr180/Tyr182), p105/NF-κB, and p50/NF-κB. GAPDH and heterogenous nuclear ribonucleoprotein L (hnRNPL) were detected as positive controls for cytosolic and nuclear fractions. (B) Control and IFIT1 shRNA-expressing THP1 cells were stimulated with 100 ng/mL LPS for the indicated times, and whole-cell lysates were subjected to western blot analysis for phospho-IRF3 (S386), phospho-STAT1 (Tyr701), IFIT1, and actin (loading control). (C) WT and Ifit1 −/− BMDM cells were stimulated with 100 ng/mL LPS for the indicated times, and whole-cell lysates were subjected to western blot analysis for phospho-STAT1 (Tyr701), phospho-p38 and MAPK (Thr180/Tyr182), phospho-JNK (Thr183/Tyr185), phospho-ERK (Thr202/Tyr204), and GAPDH (loading control). (D) WT and Ifit1 −/− mouse BMDM transfected with non-targeting control (NTC) or IFIT3 siRNA were stimulated with 100 ng/mL LPS and Ifnb1 mRNA was measured by qPCR. Data are representative of three independent experiments. Data in (D) are expressed as mean ± SD; **p < 0.01, ***p < 0.001 (two-way ANOVA followed by Sidak’s multiple comparison test). See also .

Article Snippet: Following transfer of the proteins, the nitrocellulose membrane was blocked in 5% milk for 1h and probed with the following antibodies overnight at 4°C: goat anti-IFIT1 (Santa Cruz, 82946), Rabbit anti-phos-pho-p38 (Cell Signaling, 4511S), Rabbit anti-phospho-p65 (Cell Signaling, 3033S), Rabbit anti-p105 (Santa Cruz, sc293141), Mouse anti-GAPDH (Cell Signaling, 97166), Rabbit anti-hnRNPL (Santa Cruz, sc-32317), Rabbit anti-phospho-IRF3 (Abcam, ab76493), Rabbit anti-phospho-JNK (Cell Signaling, 9255), Rabbit anti-phospho-ERK (Cell Signaling, 4370), Rabbit anti-phospho-STAT1 (Cell Signaling, 9167S).

Techniques: Control, shRNA, Expressing, Western Blot, Transfection, Comparison

Journal: Cell reports

Article Title: IFIT1 Exerts Opposing Regulatory Effects on the Inflammatory and Interferon Gene Programs in LPS-Activated Human Macrophages

doi: 10.1016/j.celrep.2018.09.002

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Following transfer of the proteins, the nitrocellulose membrane was blocked in 5% milk for 1h and probed with the following antibodies overnight at 4°C: goat anti-IFIT1 (Santa Cruz, 82946), Rabbit anti-phos-pho-p38 (Cell Signaling, 4511S), Rabbit anti-phospho-p65 (Cell Signaling, 3033S), Rabbit anti-p105 (Santa Cruz, sc293141), Mouse anti-GAPDH (Cell Signaling, 97166), Rabbit anti-hnRNPL (Santa Cruz, sc-32317), Rabbit anti-phospho-IRF3 (Abcam, ab76493), Rabbit anti-phospho-JNK (Cell Signaling, 9255), Rabbit anti-phospho-ERK (Cell Signaling, 4370), Rabbit anti-phospho-STAT1 (Cell Signaling, 9167S).

Techniques: Virus, Recombinant, Microarray, shRNA, Software