Journal: International Journal of Molecular Sciences

Article Title: The Effect of Sera from Children with Obstructive Sleep Apnea Syndrome (OSAS) on Human Cardiomyocytes Differentiated from Human Embryonic Stem Cells

doi: 10.3390/ijms222111418

Figure Lengend Snippet: NF-κB is activated by stimulation with sera from OSAS patients. ( A ) A representative picture of a cluster of cells is presented. Cardiomyocytes are identified by staining with anti-cardiac troponin (green). NF-κB subunits: Anti p50 or anti p65 (pink). Arrows point to nuclei expressing p50 or p65. ( B ) The total average intensity of NF-κB subunits p50 and p65 staining was measured in the nuclei of cardiomyocytes. The cells were incubated each time with 12 different OSAS sera and compared to 10 different control sera (5%). The results presented are an average of 3 separate experiments done in duplicates, showing a significant increase of nuclear p50 and p65 following incubation with OSAS sera. Squares and circles represent individual sera. Statistical significance was determined by the Student’s t -test comparing cells incubated with control or OSAS sera (p50 p = 0.01, p65 p = 0.001). * p < 0.05, *** p < 0.01.

Article Snippet: Rabbit anti NFκB p50 [AHP2331]: Bio-Rad Laboratories, Hercules, CA, USA.

Techniques: Staining, Expressing, Incubation, Control

Journal:

Article Title: Opposing roles of activator protein-1 and CCAAT/enhancer binding protein ? in the regulation of inducible granulysin gene expression in a human monocytic cell line, THP-1

doi: 10.1046/j.1365-2567.2002.01524.x

Figure Lengend Snippet: Suppression of granulysin promoter activity by mutation in the upstream and downstream AP-1 element, but not the NF-κB element. The upstream AP-1 binding site TGACTCA of the pGL3−329/+62 was mutated to TGAGCTC using PCR-based mutagenesis. Also, the potential NF-κB binding site GGGGTTTCTCC of the pGL3−329/+62 or pGL3−329/+62 AP-1 mutant was mutated to TACCTTTCTCC using PCR-based mutagenesis. In addition, the downstream AP-1 binding site CCTGACCTGCT of the pGL3−193/+62 was mutated to CCGAGCTCGCT using PCR-based mutagenesis. THP-1 cells (4 × 105 cells) were transfected with various truncated or mutated promoter constructs, as described in the Materials and Methods. After 24 hr, transfected cells were incubated with or without 10 μg/ml A. laidlawii for 24 hr. Then the cells were lysed and assayed for luciferase activity. Data are presented as in the legend to Fig. 1. *P < 0·05; **P < 0·01.

Article Snippet: Rabbit anti-NF-κB p50 pAb was obtained from Rockland (Gilbertsville, PA).

Techniques: Activity Assay, Mutagenesis, Binding Assay, Transfection, Construct, Incubation, Luciferase

Journal:

Article Title: Opposing roles of activator protein-1 and CCAAT/enhancer binding protein ? in the regulation of inducible granulysin gene expression in a human monocytic cell line, THP-1

doi: 10.1046/j.1365-2567.2002.01524.x

Figure Lengend Snippet: EMSA of putative transcription factor binding sites in the upstream region of the granulysin gene. The following oligonucleotides were used: (a) NF-κB (−221/−197), 5′-GTACTAAGGGGTTTCTCCCTCCATC-3′ (−221 to −197 bp); (b) AP-1 (−109/−84), 5′-GGGCTACCACTGCCCTGACCTGCTTC-3′ (−109 to −84 bp); and (c) C/EBPβ (−1008/−986), 5′-GGCCAACATGGTGAAACCCTGTC-3′ (−1008 to −986 bp). A 32P-labelled, double-stranded oligonucleotide probe was incubated with nuclear extracts from THP-1 cells stimulated with or without 10 μg/ml A. laidlawii for 24 hr, as described in the Materials and Methods. The competitors were used in a 10- or 100-fold molar excess over labelled probes. −279/−260, −221/−197, −109/−84 and −1008/−986 indicate unlabelled probes. Supershift assays were perfomed with 1 μg of appropriate antibodies: rabbit anti-c-Jun/AP-1 antibody (c-Jun), rabbit anti-NF-κB p50 antibody (p50), rabbit anti-NF-κB p65 antibody (p65), and rabbit anti-C/EBPβ antibody (C/EBPβ) or normal rabbit IgG as a cotrol (IgG). The asterisk (*) indicates nuclear extract from A. laidlawii-stimulated THP-1 cells.

Article Snippet: Rabbit anti-NF-κB p50 pAb was obtained from Rockland (Gilbertsville, PA).

Techniques: Binding Assay, Incubation

Journal:

Article Title: Rapid Protection of Gnotobiotic Pigs against Experimental Salmonellosis following Induction of Polymorphonuclear Leukocytes by Avirulent Salmonella enterica

doi: 10.1128/IAI.71.4.2182-2191.2003

Figure Lengend Snippet: Western blotting analysis of INT 407 whole-cell lysates probed with anti-NF-κB p50 antibody following incubation of monolayers with S. enterica serovar Typhimurium F98 Nalr (F98) or rough S. enterica serovar Infantis (S.inf.φr) at 37°C for 6 h (MOI = 10). +ve control, 1 μg of PMA/ml; −ve control, INT 407 cells incubated in RPMI 1640 medium without Salmonella or PMA; NFκB, p50 protein.

Article Snippet: The nitrocellulose was then incubated with rabbit anti-mouse NF-κB (p50) (1:200) (Autogenbioclear; Calne, Wilts, United Kingdom) for 60 min at room or ambient temperature on an end-to-end shaker.

Techniques: Western Blot, Incubation

Journal: Cell Communication and Signaling : CCS

Article Title: Knockdown of trem2 promotes proinflammatory microglia and inhibits glioma progression via the JAK2/STAT3 and NF-κB pathways

doi: 10.1186/s12964-024-01642-6

Figure Lengend Snippet: Effect of trem2 on STAT1 phosphorylation and the NF-κB p50 signaling pathway, as well as animal studies. (a) Western blot analysis of itgam in HMC3 and BV2 cells. (b-c) Western blot analysis of anti-inflammatory cytokines (IL-10) and pro-inflammatory cytokines (IL-1β, IL-6, TNF-α). (d) Western blot analysis of NF-κB p50 in microglia. (e) Western blot analysis of jak2 and p-jak2 in microglia. (f) Western blot analysis of stat3 and p-stat3 in microglia. (g) Western blot analysis of stat1 and p-stat1 in microglia. (h) Representative image of intracranial tumor of mice ( n = 8 of each group). (i) The representative IHC images of IBA1. (j) The representative IF images of CD105. (k) The representative IF images of Ki-67. (l) The representative IHC images of γH 2 ax

Article Snippet: Primary antibodies included rabbit anti-trem2 (1:500, Abcam, ab209814), rabbit anti-bax (1:1000, Proteintech, Wuhan, China, 50599-2-Ig), rabbit anti-bcl-2 (1:1000, Proteintech, 12789-1-AP), rabbit anti-caspase-3 (1:1000, Abcam, ab184787), rabbit anti-cd11b (1:1000, Abcam, ab133357), mouse anti-gapdh (1:40000, Proteintech, 60004-1-Ig), rabbit anti-γH 2 ax (1:5000, Abcam, ab81299), rabbit anti-il-1β (1:500, Bioss, Beijing, bs-0812R), rabbit anti-il-6 (1:500, Bioss, bs-6309R), rabbit anti-il-10 (1:1000, Proteintech, 20850-1-AP), rabbit anti-iNOS (1:500, Affinity, Changzhou, China, AF0199), rabbit anti-jak2 (1:500, Proteintech, 17670-1-AP), rabbit anti-NF-κB p105/p50 (1:2000, Abmart, Shanghai, M005873), rabbit anti-Phospho-jak2 (1:500, Abmart, T56570), rabbit anti-cleaved-caspase-3 (1:500, Abmart, TA7022), rabbit anti-Phospho-stat1 (1:500, Abmart, TP56498), rabbit anti-Phospho-stat3 (1:500, Affinity, AF3293), rabbit anti-stat1 (1:500, Abmart, T55227), mouse anti-stat3 (1:1000, Proteintech, 60199-1-Ig), rabbit anti-TNF Alpha (1:500, Proteintech, 17590-1-AP), mouse anti-vegfa (1:1000,Preteintech, 66828-1-Ig), rabbit anti-ATP1A1 (1:5000, Proteintech, 14418-1-AP).

Techniques: Western Blot