Journal: Leukemia

Article Title: A point mutation of zebrafish c-cbl gene in the ring finger domain produces a phenotype mimicking human myeloproliferative disease

doi: 10.1038/leu.2015.154

Figure Lengend Snippet: Increase in HSPCs was dependent on the Flt3 pathway. (a–d) flt3 MO injection attenuated the phenotype of LDD731. (e and f) c-myb staining of zebrafish embryos after treatment with 500 nM Lestaurtinib. (g and h) c-myb staining of embryos after treatment with 100 nM PKC412. (i) CBL mutation decreased FLT3 ubiquitination levels and upregulated mature FLT3. 293T cells were transfected with FLT3 and/or c-CBL constructs as indicated. After lysis, FLT3 was immunoprecipitated, blotted and probed with anti-FLT3 and anti-FK2. (j) Inhibitors were used to treat 293T cells. The cells transfected with FLT3 and c-CBL were treated with or without PKC412 or Lestaurtinib for 24 h. After lysis, protein samples were blotted and probed with FLT3, c-CBL and pFLT3 antibodies.

Article Snippet: FLT3 (fms-like tyrosine kinase 3) inhibitor PKC412 (ref. 22 ) (sc-200691) and Lestaurtinib 23 (sc-218657) with stock concentrations of 10 mM, were purchased from Santa Cruz Biotechnology and stored at − 80 °C.

Techniques: Injection, Staining, Mutagenesis, Ubiquitin Proteomics, Transfection, Construct, Lysis, Immunoprecipitation

Journal: Marine Drugs

Article Title: Pseudopterosin A-D Modulates Dendritic Cell Activation in Skin Sensitization

doi: 10.3390/md23060245

Figure Lengend Snippet: Pseudoperosin (Ps)A-D reduces the nickel sulfate (NiSO 4 ) induced upregulation of DC activation markers CD54 and CD86 in a similar manner as dexamethasone. THP-1-derived dermal dendritic cell surrogates (DDCs) were seeded with 1 × 10 6 cells/mL into a 24-well plate. Cells were pre-treated with dexamethasone [1 µM] or PsA-D [20 µM] for 1 h, followed by NiSO 4 [380 µM] treatment for 23 h. Surface marker analysis was performed via flow cytometry and is depicted as histograms ( A ) and percentage of positive cells ( B ). Error bars indicate the standard errors of the mean ( n = 3 independent experiments with * = p ≤ 0.05 and ** = p ≤ 0.01).

Article Snippet: Cells were stained with the following antibodies (diluted 1:50) for 10 min at 4 °C in the dark: REA Control (S)-APC (Miltenyi Biotec, #130-113-434), CD54-APC (Miltenyi Biotec, #130-121-342), and CD86-APC (Miltenyi Biotec, #130-116-161).

Techniques: Activation Assay, Derivative Assay, Marker, Flow Cytometry

Journal: Frontiers in Molecular Neuroscience

Article Title: Amyloid β1-42 (Aβ1-42) Induces the CDK2-Mediated Phosphorylation of Tau through the Activation of the mTORC1 Signaling Pathway While Promoting Neuronal Cell Death

doi: 10.3389/fnmol.2017.00229

Figure Lengend Snippet: Aβ facilitates HIF1α synthesis and autophagy inhibition via mTOR activation. (A) SK-N-MC cells were exposed to Aβ (5 μM) for 0–48 h. HIF1α and β-actin expression was analyzed by western blot. n = 3. (B) Cells were pretreated with NAC (1 mM) for 30 min prior to Aβ treatment for 24 h. HIF1α and β-actin expression were analyzed by western blot. n = 3. (C,E) Cells were incubated with rapamycin (10 nM) for 30 min prior to Aβ treatment for 24 h. Phosphorylation of 4EBP1 (Thr 37/46) and 4EBP1, phosphorylation of p70S6K1 (Thr 389), HIF1α and β-actin were analyzed by western blot. n = 6. (D) Protein samples were immunoprecipitated by eukaryotic translation initiation factor 4E (eIF4E) antibody-conjugated protein A/G agarose beads. Samples were blotted with 4EBP1 and eIF4E-specific antibodies. n = 3. (F) Cells were exposed to PF4708671 (10 μM) for 30 min prior to Aβ treatment for 24 h. HIF1α and β-actin expression was detected by western blot. n = 6. (G) Cells were exposed to cycloheximide (4 μM) for 30 min prior to Aβ treatment for 24 h. HIF1α and β-actin expressions were detected by western blot. n = 6. (H) Cells were pretreated with rapamycin (10 nM) for 30 min, incubated with Aβ for 24 h and analyzed by western blotting with LC3, p62 and β-actin specific antibodies. n = 3–6. (I) LC3 puncta was visualized by confocal microscopy. Presented results are merged images. Green and red fluorescents indicate LC3 and PI respectively. Scale bars, 50 μm (magnification × 600). (J) Cells were pretreated with trehalose (10 μM) for 30 min prior to Aβ treatment for 24 h. Cytotoxicity was measured by MTT assay at an absorbance of 545 nm using a microplate reader. Data present the mean ± SE. n = 6. (K) Cell viability was measured by trypan blue exclusion assay. Data are presented as a mean ± SE. n = 6. Each blot image was presented as representative image. * p < 0.05 vs. control, # p < 0.05 vs. Aβ treatment.

Article Snippet: The antibodies of hypoxia inducible factor (HIF1α; NB100-105), p70S6K1 (NB600-1049), LC3 (NB100-2220) and p62 (NBP1-48320) were obtained from Novus Biologicals (Littleton, CO, USA) and the HRP-conjugated goat anti-rabbit IgG was purchased from Santa Cruz Biotechnology.

Techniques: Inhibition, Activation Assay, Expressing, Western Blot, Incubation, Phospho-proteomics, Immunoprecipitation, Confocal Microscopy, MTT Assay, Trypan Blue Exclusion Assay, Control