Journal: The Journal of biological chemistry

Article Title: Identification of signal-induced IkappaB-alpha kinases in human endothelial cells.

doi: 10.1074/jbc.271.33.19680

Figure Lengend Snippet: FIG. 6. IkB-a kinases specifically phosphorylate IkB-a but not c-Jun or the p50 or p65 subunits of NF-kB. Cytoplasmic extracts were prepared from HUVEC monolayers stimulated with TNFa (300 units/ml) for 3 min. IkB-a kinases were purified from cytoplasmic extracts by an IkB-a-affinity method as described under “Materials and Methods.” Purified kinases were incubated with IkB-a (lane 3), p50 (lane 4), p65 (lane 5), MBP (lane 6), or c-Jun (lane 7) in a kinase reaction as described under “Materials and Methods.” As controls, kinase reac- tions were performed with IkB-a alone (lane 1) and with purified ki- nases alone (lane 2). A, Coomassie-stained SDS-PAGE gel. B, autora- diograph of A.

Article Snippet: Peptide-specific rabbit polyclonal antibodies against IkB-a, p50, and p65 were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Techniques: Purification, Incubation, Staining, SDS Page

Journal: Microbes and infection

Article Title: CD38 deficiency up-regulated IL-1β and MCP-1 through TLR4/ERK/NF-κB pathway in sepsis pulmonary injury.

doi: 10.1016/j.micinf.2021.104845

Figure Lengend Snippet: Figure 4. The protein levels of NF-κB and phosphorylation of NF-κB. The protein levels 579

Article Snippet: The membrane was washed by TBST according to the manufacturer’s 164 instructions and incubated with anti-NF-κB p65 Rabbit mAb (1:1000) (CST, USA), 165 anti-Phospho-NF-κB p65 Rabbit mAb (1:500) (CST, USA), anti-NF-κB1 p105/p50 Rabbit 166 mAb (1:1000) (CST, USA), anti-Phospho-NF-κB p105 Rabbit mAb (1:500) (CST, USA), 167 anti-MAPK ERK1/2 Rabbit mAb (1:2000) (CST, USA), anti-Phospho-MAPK ERK1/2 168 Rabbit mAb (1:2000) (CST, USA), anti-MAPK JNK Rabbit mAb (1:2000) (CST, USA), 169 anti-Phospho-MAPK JNK Rabbit mAb (1:2000) (CST, USA), anti-MAPK p38 Rabbit mAb 170 (1:2000) (CST, USA), anti-Phospho-MAPK p38 Rabbit mAb (1:2000) (CST, USA), 171 anti-iNOS Rabbit mAb (1:1000) (CST, USA),anti-IL-1β Rabbit mAb (1:500) (Boster, China), 172 Jo urn al Pr e-p roo f anti-TNF-α Rabbit mAb (1:500) (Boster, China), anti-CCR2 Rabbit mAb (1:1000) (CST, 173 USA),anti-MCP-1 Rabbit mAb (1:1000) (CST, USA), and anti-GAPDH mAb (1:5000) 174 (Proteintech, USA).

Techniques: Phospho-proteomics

Journal: Stem cell research & therapy

Article Title: Intracellular osteopontin potentiates the immunosuppressive activity of mesenchymal stromal cells.

doi: 10.1186/s13287-024-03979-8

Figure Lengend Snippet: Fig. 5 iOPN promoted STAT1-mediated immunosuppression in MSCs. a–d Vector-MSCs and iOPN-MSCs or WT-MSCs and OPN−/−-MSCs were treated with or without TNF-α plus IFN-γ (10 ng/mL) for the indicated times. Cells were harvested, and OPN, NF-κB p65, STAT1, IKBα, phosphorylation of NF-κB p65, phosphorylation of STAT1 at Tyr701 and phosphorylation of IKBα were analyzed by immunoblotting analysis. Full-length blots are presented in Additional file 1: Fig. 5a-d. e–g Vector-MSCs and iOPN-MSCs were stimulated with TNF-α plus IFN-γ (10 ng/mL) for 12 h. The STAT1 inhibitor fludarabine (Flu, 2 μM) was then added to the culture medium of Vector-MSCs and iOPN-MSCs. The expression of iNOS was determined by quantitative real-time PCR (e), immunoblotting analysis (f) and flow cytometry (g). Full-length blots are presented in Additional file 1: Fig. 5f. h Vector-MSCs and iOPN-MSCs were pretreated with DMSO or fludarabine (Flu, 2 μM) for 6 h and then irradiated and cocultured with CFSE-labeled splenocytes activated by anti-CD3/CD28 antibodies for 3 days at a ratio of 1:20. CD4+ T cells and CD8+ T cells were stained for proliferation analysis by flow cytometry at the end of coculture, and the percentages of proliferating T cells are shown. The results are representative of three to six independent experiments. Values are shown as the mean ± SEM and statistical significance is indicated as *P < 0.05, **P < 0.01 and ***P < 0.001

Article Snippet: Antibodies against GAPDH (CAT# 2118, RRID: AB_561053), NF-κB p65 (CAT# 8242, RRID:AB_10859369), phospho-NF-κB p65 (CAT# 3033, RRID:AB_331284), STAT1 (CAT# 14994, RRID:AB_2737027), phospho-STAT1 (Tyr701) (CAT# 7649, RRID:AB_10950970), IκBα (CAT# 4814, RRID:AB_390781), phospho-IκBα (CAT# 2118) and ubiquitin (CAT# 3936, RRID: RRID:AB_331292) were purchased from Cell Signaling Technology (Danvers, Massachusetts, USA).

Techniques: Plasmid Preparation, Phospho-proteomics, Western Blot, Expressing, Real-time Polymerase Chain Reaction, Flow Cytometry, Irradiation, Labeling, Staining

Journal: Annals of the rheumatic diseases

Article Title: Leptin produced by joint white adipose tissue induces cartilage degradation via upregulation and activation of matrix metalloproteinases.

doi: 10.1136/annrheumdis-2011-200372

Figure Lengend Snippet: Figure 2 Signalling pathways activated after leptin and leptin plus IL-1 stimulation of chondrocytes and their role in collagenase expression. Primary human articular chondrocytes were serum starved overnight and then stimulated for 20 and 60 min with either leptin (25 µg/ml) or IL-1 (0.25 ng/ml) alone or in combination. Cells were lysed, and extracts immunoblotted with antibodies against (A) phospho-STAT1 (Y701 or S727), phospho-STAT3 (Y705 and S727), phospho-STAT5 (Y694) and β-tubulin as loading control; (B) phospho-Erk (p44/42) (T202/Y204 of Erk1), phospho-JNK (SAPK) (T183/Y185), phospho-p38 (T180/Y182); or (C) phospho-Akt (S473 and Y308) and phospho-p65 (S536). (D and E) Primary human chondrocytes were stimulated with leptin (25 µg/ml) with or without the inhibitors, LY (LY249002, 5 µM), U0126 (5 µM), SB (SB203580, 10 µM), SP (SP600125, 10 µM), Akt IV (3 µM) or Akt VIII (3 µM) for 20 h. Cells were pretreated for 30 min with inhibitors or vehicle control before the addition of leptin. The expression of MMP1 (D) and MMP13 (E) was assayed by real-time reverse transcription PCR and expressed as fold relative to the control after normalisation to 18s rRNA. *p<0.05; **p<0.01; ***p<0.001 versus control. Data are representative of three separate experiments. IL, interleukin; MMP, matrix metalloproteinase.

Article Snippet: Primary antibodies against phospho-Erk1/2 (phospho-p44/42) (#9101), phospho-p38 (#9211), phosphoJNK1/2 (#9251), phospho-p65 (Ser 536; #3031) and phosphoSTAT were all from Cell Signaling Technology (New England Biolabs, Hitchin, UK).

Techniques: Expressing, Control, Reverse Transcription