Journal: Journal of Cell Science

Article Title: Phosphorylation of the RNA-binding protein Zfs1 modulates sexual differentiation in fission yeast

doi: 10.1242/jcs.208066

Figure Lengend Snippet: Nitrogen depletion and inhibition of TOR signaling promote Zfs1 phosphorylation . (A) Western blot of Zfs1 protein during incubation in medium without nitrogen. Cells were grown in minimal EMM to mid-log phase, then washed with medium without nitrogen and incubated in this for the indicated times. (B) Protein extracts from cells treated as in A were incubated with λ-protein phosphatase (see Materials and Methods). (C) Torin-1 inhibition of TOR complexes stimulates Zfs1 hyper-phosphorylation. Cells growing in EMM were incubated with 10 µM Torin-1 or the same volume of DMSO for the indicated times. (D) The AKT1-related Gad8 kinase is required for hyper-phosphorylation of Zfs1. Cells were grown in EMM and then washed and incubated in EMM without nitrogen for 2 h.

Article Snippet: For protein dephosphorylation assays, up to 150 µg of protein extracted with beating buffer without EDTA and phosphatase inhibitors, was treated with 400 U of λ-protein phosphatase (New England Biolabs) for 50 min at 30°C.

Techniques: Inhibition, Western Blot, Incubation

Journal: Pharmaceutical Research

Article Title: Photochemically Controlled Drug Dosing from a Polymeric Scaffold

doi: 10.1007/s11095-017-2164-9

Figure Lengend Snippet: Overlaid representative UV-vis spectra of ( a ) 2 and ( b ) 3 in acetonitrile (2 x 10 −5 M) after various periods of 365 nm irradiation. Arrows indicate absorbance trends with time and irradiation times used were 0, 30, 60, 90,120,150, 240, 300, 360,450, 570, and 690 s, respectively.

Article Snippet: UV-visible absorption spectra were collected between 200–400 nm using a Perkin Elmer Lambda 650 UV-visible spectrophotometer interfaced with UV WinLab software at recorded time intervals during the irradiation period.

Techniques: Irradiation

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: NDR2 kinase contributes to cell invasion and cytokinesis defects induced by the inactivation of RASSF1A tumor-suppressor gene in lung cancer cells

doi: 10.1186/s13046-019-1145-8

Figure Lengend Snippet: NDR2 interacts and phosphorylates GEF-H1 in HBEC. HBEC-3 or H1299 cells were transfected with siNeg and/or siRASSF1A, siNDR1, siNDR2, siGEF-H1, pcDNA3-NDR1, pcDNA3-NDR2 or pcB6-GEF-H1. a GST-RBD pull-down assay in H1299 cells. b Ser885 phosphorylation from GEF-H1 assayed by western blot following λ-phosphatase pre-treatment of the total protein extract or not and normalized with total GEF-H1 expression in HBEC-3 cells. c GST-NDR1 or -NDR2 pull-down assay using siRNA & GEF-H1 plasmid as controls. NDR2 phosphorylation on Ser265-GEFGH1 link with phosphorylation on Ser-885-GEF-H1A was assayed. NDR2 activity on HBEC-3 cells extracts previously transfected with pcB6-GEF-H1 wild type, mutated on Ser265 (S265A), or on Ser885 (S885A) was assayed by quantifying ser885-GEF-H1 phosphorylation status by western blot following normalization by total GEF-H1 expression. Error bars indicate the SEM ( n ≥ 3). * P < 0.05, ** P < 0.01 and *** P < 0.001, using an ANOVA test followed by Dunnett’s test

Article Snippet: Proteins (2 μg) were incubated with 400 units of λ-phosphatase (Santa Cruz™) and 2 mM MnCl2 at 30 °C.

Techniques: Transfection, Pull Down Assay, Western Blot, Expressing, Plasmid Preparation, Activity Assay