Journal: Cells

Article Title: NFκB- and MAP-Kinase Signaling Contribute to the Activation of Murine Myeloid Dendritic Cells by a Flagellin A: Allergen Fusion Protein

doi: 10.3390/cells8040355

Figure Lengend Snippet: Both MAPK- and NFκB-signaling contribute to rFlaA:Betv1-induced mDC activation while only JNK MAPK-signaling contributes to mTOR-dependent phosphorylation of p70 S6 kinase: 1 × 10 6 BALB/c mDCs were pre-treated for 3 h with triptolide (T, 10 nM), SB202190 (S, 60 µM), U0126 (U, 10 µg/mL), dexamethason (D, 40 ng/mL), TPCA-1 (TC, 60 nM), BMS-345541 (B, 1 µM), or SP600125 (SP, 25 µM) washed, and subsequently stimulated for 30 min with the indicated proteins. Target proteins in lysates were detected by Western Blot . Data are representative or mean results of two to five independent experiments ± SD.

Article Snippet: Target proteins in lysates were detected by Western Blot using the iBind System (Thermo Fisher Scientific, Darmstadt, Germany) and antibodies from Cell Signaling Technologies (Leiden, The Netherlands): mTOR regulation antibody sampler kit, mTOR substrates antibody sampler kit, NFκB pathway sampler kit, MAPK family antibody sampler kit, loading control antibody sampler kit (HRP Conjugate).

Techniques: Activation Assay, Western Blot

Journal: Cells

Article Title: NFκB- and MAP-Kinase Signaling Contribute to the Activation of Murine Myeloid Dendritic Cells by a Flagellin A: Allergen Fusion Protein

doi: 10.3390/cells8040355

Figure Lengend Snippet: Suggested mechanism of rFlaA:Betv1-mediated mDC activation. Stimulation of mDCs with rFlaA:Betv1 results in enhanced uptake of the fusion protein and a mammalian target of rapamycin (complex 1) (mTORC1)-dependent activation of mDC metabolism and immune modulatory IL-10 secretion, likely mediated by hypoxia-inducible factor 1-alpha (HIF-1α)- and avian myelocytomatosis virus oncogene cellular homology (c-Myc)- and signal transducer/activator of transcription 3 (STAT-3)-signaling, respectively (1). rFlaA:Betv1 also triggers a myeloid differentiation primary response 88 (MyD88)-dependent, interleukin-1 receptor-associated kinase 1 (IRAK1)/IRAK4/TNF receptor associated factor 6 (TRAF6)/transforming growth factor beta-activated kinase 1 (TAK1)-mediated activation of the MAP kinase signaling pathway (2). Once activated, MAPK-signaling can cross-trigger activation and nuclear translocation of NFκB via TAK1-mediated activation of IKKα (3). Here, both NFκB- (promoting IL-12 secretion) and MAP kinase-signaling (promoting IL-6 and TNF-α secretion) contribute to rFlaA:Betv1-induced pro-inflammatory cytokine secretion. In addition, the activation of MAP kinase signaling can cross-activate the mTOR pathway by either inhibiting tuberous sclerosis complex 1/2 (TSC-1/TSC-2) complexes, phosphorylation of regulatory associated protein of mTOR (RAPTOR), or direct activation of mTOR downstream targets like p70 S6 kinase by ERK/ribosomal S6 kinase (RSK) (4). The MAPK-dependent mTOR activation also contributes to IL-10 secretion while contributing to rFlaA:Betv1-induced activation of mDC metabolism via JNK MAPK activation. The different inhibitors used in this study are indicated in orange, orange arrows depict inhibitory signals, black arrows depict activating signals.

Article Snippet: Target proteins in lysates were detected by Western Blot using the iBind System (Thermo Fisher Scientific, Darmstadt, Germany) and antibodies from Cell Signaling Technologies (Leiden, The Netherlands): mTOR regulation antibody sampler kit, mTOR substrates antibody sampler kit, NFκB pathway sampler kit, MAPK family antibody sampler kit, loading control antibody sampler kit (HRP Conjugate).

Techniques: Activation Assay, Translocation Assay

Journal: Oncotarget

Article Title: Tubeimoside-1 suppresses tumor angiogenesis by stimulation of proteasomal VEGFR2 and Tie2 degradation in a non-small cell lung cancer xenograft model.

doi: 10.18632/oncotarget.6676

Figure Lengend Snippet: Figure 5: a. Western blot analysis of VEGFR2, Tie2, p-AKT/AKT and p-mTOR/mTOR expression of eEND2 cells (% of control at each time point), which were treated for 0.5h (white circles), 1h (light grey circles), 2h (dark grey circles) and 3h (black circles) with vehicle (0μM; control), 5 and 10μM TBMS1. The data were quantified from 3 independent experiments. Means ± SEM. *P<0.05 vs. vehicle. b. Western blot analysis of VEGFR1, Tie1, VEGFR2 and Tie2 expression of eEND2 cells (% of control), which were treated for 3h with vehicle (0μM; control) or 10μM TBMS1. The data were quantified from 3 independent experiments. Means ± SEM. *P<0.05 vs. vehicle.

Article Snippet: After blockade of non-specific binding sites, membranes were incubated overnight at 4°C with a rabbit monoclonal anti-VEGFR2 antibody (1:500; Cell Signaling Technology), a goat polyclonal anti-Tie2 antibody (0.3μg/mL; R&D Systems), a rabbit polyclonal anti-phosphorylated (p)-AKT1/2/3 antibody (Thr308; 1:100; Santa Cruz Biotechnology, Heidelberg, Germany), a rabbit monoclonal anti-AKT antibody (1:500; Cell Signaling Technology), a rabbit monoclonal anti-p-mTOR antibody (Ser2448; 1:500; Cell Signaling Technology), a rabbit monoclonal anti-mTOR antibody (1:500; Cell Signaling Technology), a rabbit polyclonal antiVEGFR1 antibody (1:100; Santa Cruz Biotechnology), a rabbit polyclonal anti-Tie1 antibody (1:100; Santa Cruz Biotechnology) or a mouse monoclonal anti-βactin antibody (1:2,000, Sigma-Aldrich) followed by the corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies (1:3,000; GE Healthcare, Freiburg, Germany).

Techniques: Western Blot, Expressing, Control