Journal: Nature Communications

Article Title: Reduced efficacy of a Src kinase inhibitor in crowded protein solution

doi: 10.1038/s41467-021-24349-5

Figure Lengend Snippet: Spatial distribution functions (SDF) of a PP1 and b BSA around c-Src kinase. In the case of BSA-SDF, Cα atoms are used for analysis. The PP1-SDF is shown as isosurface at 0.5% (transparent) and 1.5% occupancies (solid). For BSA-SDF, the isosurface at 1.0% occupancy is shown.

Article Snippet: In brief, purified Src protein (20 ng) was incubated with the indicated concentrations of PP1 (Cayman Chemical), 100 μg/ml Src substrate KVEKIGEGTYGVVYK-amide (SignalChem Pharmaceuticals), and 10 μM ATP for 60 min at room temperature in a reaction solution containing 0.2% DMSO, 40 mM Tris-HCl (pH7.5), 20 mM MgCl 2 , 2 mM MnCl 2 , 50 μM DTT with and without 5.0 mg/ml BSA.

Techniques:

Journal: Nature Communications

Article Title: Reduced efficacy of a Src kinase inhibitor in crowded protein solution

doi: 10.1038/s41467-021-24349-5

Figure Lengend Snippet: a TYR-in state structure together with a binding trajectory in the dilute solution. b TYR-out state structure with binding trajectory in the dilute and crowded solution (Src8BSA). Red, blue, and green spheres represent the PP1 bound position in the crystal structure, representative binding pathways in dilute and crowded solutions, respectively. Encounter states in the pathways are circled. c Distribution for Tyr82-Gly86 distance, g ( d ). The distance between Tyr82 and Gly86 in a crystal structure of c-Src kinase in a complex with an inhibitor (ID: 1Y57) is shown with dashed line for comparison .

Article Snippet: In brief, purified Src protein (20 ng) was incubated with the indicated concentrations of PP1 (Cayman Chemical), 100 μg/ml Src substrate KVEKIGEGTYGVVYK-amide (SignalChem Pharmaceuticals), and 10 μM ATP for 60 min at room temperature in a reaction solution containing 0.2% DMSO, 40 mM Tris-HCl (pH7.5), 20 mM MgCl 2 , 2 mM MnCl 2 , 50 μM DTT with and without 5.0 mg/ml BSA.

Techniques: Binding Assay

Journal: Stem Cells Translational Medicine

Article Title: Differentiation and Characterization of Dopaminergic Neurons From Baboon Induced Pluripotent Stem Cells

doi: 10.5966/sctm.2015-0073

Figure Lengend Snippet: Progression of baboon fibroblast-derived induced pluripotent stem cells (biPSCs) to fully differentiated neurons. (A): Summary graph of the differentiation process. (B): Colony of baboon induced pluripotent stem cells before differentiation. (C): Colony of biPSCs after 8 days of differentiation with neural rosettes (arrowheads). (D): Colony of biPSCs after 15 days of differentiation just before passaging. Neural rosettes have expanded and the colony was fully confluent. (E): Neural precursor cells after passage. (F): At day 32, cells expressed early neuronal marker NESTIN. (G): At day 32, few cells expressed mature neuronal marker βIII-TUBULIN. (H, J): As the cells differentiate, they adopt a morphology with larger soma and increased branching and length of neuronal processes. This is shown in higher-magnification images (I) and (K) (arrows). All scale bars = 100 µm. Abbreviations: BDNF, brain-derived neurotrophic factor; DAPI, 4′,6-diamidino-2-phenylindole; FGF8, fibroblast growth factor 8; GDNF, glial cell-derived neurotrophic factor; KSR, knockout serum replacer; SAG, smoothened agonist; SHh, sonic hedgehog.

Article Snippet: Neurons were then matured for 2 weeks in DMEM/F12 containing 1% N2 supplement and 2% B27 supplement (Thermo Fisher Scientific Life Sciences), 10 ng/ml BDNF (Peprotech), 20 ng/ml of glial cell-derived neurotrophic factor (GDNF; Peprotech), 0.5 mM dibutyryl cyclic adenosine monophosphate (cAMP; Sigma-Aldrich), 0.2 mM ascorbic acid (Sigma-Aldrich), 2 mM GlutaMax, 100 U/ml penicillin, and 100 μg/ml streptomycin.

Techniques: Derivative Assay, Passaging, Marker, Knock-Out