Journal: The Febs Journal

Article Title: ENPP 1 processes protein ADP ‐ribosylation in vitro

doi: 10.1111/febs.13811

Figure Lengend Snippet: Production of ENPP recombinant proteins. (A) Representative and schematic illustrations of recombinant mouse ENPP 2‐1‐Target (m ENPP 2‐1‐T) chimera purified as described in Kato et al . [42,43] and recombinant 8xHistidine tag version (m ENPP 2‐1‐8xHis). (B) Flow chart for the purification of m ENPP 2‐1‐T and m ENPP 2‐1‐8xHis. (C) Left panel, the purity of recombinant m ENPP 2‐1‐T and m ENPP 2‐1‐8xHis enzymes was analysed using separation of 20 μ m of protein on an SDS / PAGE gel followed by staining with Coomassie. Right panel, 20 μ m of m ENPP 2‐1‐T was probed by anti‐6xHis western blot. (D) 1 μg of m ENPP 2‐1‐T and m ENPP 2‐1‐8xHis enzymes were used as substrates for PNG ase F and Endo H deglycosylation enzymes. Samples were resolved on SDS / PAGE and stained by Coomassie. Black star indicates glycosylated ENPP 1, green star indicates deglycosylated ENPP 1, red star indicates deglycosylation resistant ENPP1.

Article Snippet: The 6xHis‐ hs PARP1 (wild‐type) was expressed and purified from E. coli , attached to MagneHis beads (Promega Corporation, Madison, WI, USA), and PARylated as described previously with the following changes: PARP1 (final concentration 1 μ m ) was autoPARylated in the presence of 1 m m β‐NAD + for 30 min at 37 °C.

Techniques: Recombinant, Purification, SDS Page, Staining, Western Blot

Journal: The Febs Journal

Article Title: ENPP 1 processes protein ADP ‐ribosylation in vitro

doi: 10.1111/febs.13811

Figure Lengend Snippet: ENPP 1 is able to hydrolyse protein poly( ADP ‐ribosyl)ation. About 70 n m of human recombinant PARP 1 was automodified to produce ~ 3 μ m PAR substrate (defined in monomeric ADP ‐ribose units) and incubated with buffer only (control) and decreasing concentrations of m ENPP 2‐1‐T (A), m ENPP 2‐1‐8xHis (B) and NUDT 16 (C). Samples were fractionated on SDS / PAGE and transferred on nitrocellulose membranes. Membranes were first stained with S‐Ponceau and then probed with anti‐ PAR antibody. (D) Time point hydrolysis of PAR ylated PARP 1 was performed at indicated concentrations and times with NUDT 16, m ENPP 2‐1‐T and SVP . Samples were resolved on SDS / PAGE and transferred on nitrocellulose membranes. Membranes were first stained with S‐Ponceau and then probed with anti‐ PAR antibody.

Article Snippet: The 6xHis‐ hs PARP1 (wild‐type) was expressed and purified from E. coli , attached to MagneHis beads (Promega Corporation, Madison, WI, USA), and PARylated as described previously with the following changes: PARP1 (final concentration 1 μ m ) was autoPARylated in the presence of 1 m m β‐NAD + for 30 min at 37 °C.

Techniques: Recombinant, Incubation, SDS Page, Staining

Journal: The Febs Journal

Article Title: ENPP 1 processes protein ADP ‐ribosylation in vitro

doi: 10.1111/febs.13811

Figure Lengend Snippet: ENPP 1 is able to hydrolyse protein poly‐ and mono‐( ADP ‐ribosyl)ation producing PRAMP and AMP . (A) Schematic illustration of protein ADP ‐ribosylation in the presence of NAD + labelled on the alpha phosphate group with 32 P. Enzymes used in experiments showed in panels B–D, and cleavable chemical bonds in radiolabelled MAR / PAR were indicated. Main reaction products of phosphodiesterases‐dependent hydrolysis of radiolabelled protein PAR ylation were represented. (B) Human recombinant PARP 1 was automodified in the presence of [ 32 P]‐ NAD + and then incubated with buffer (Control), 18 μ m of recombinant NUDT 16, 4 μ m of recombinant ENPP 2‐1‐T, 1 μ m of PARG and 0.45 μ m of purified SVP for 3 h at 30 °C. In top panel, samples were resolved on SDS / PAGE and [ 32 P]‐ NAD + incorporation was detected by autoradiography. In bottom panel, reactions described in top panel were loaded on TLC plate. (C) Top panel, 1 μ m of recombinant PARP 1‐E988Q mutant was automodified using 32 P‐labelled NAD + and then incubated with buffer only (control), 5 μ m of recombinant NUDT 16, 5 μ m of recombinant m ENPP 2‐1‐T or 2 μ m of purified SVP . Samples were resolved on SDS / PAGE and [ 32 P]‐ NAD + incorporation was detected by autoradiography. Bottom panel, 1 μ m of recombinant GST ‐ PARP 10cd was automodified and treated as indicated in top panel. (D) The products of indicated enzymatic reactions were assayed by TLC.

Article Snippet: The 6xHis‐ hs PARP1 (wild‐type) was expressed and purified from E. coli , attached to MagneHis beads (Promega Corporation, Madison, WI, USA), and PARylated as described previously with the following changes: PARP1 (final concentration 1 μ m ) was autoPARylated in the presence of 1 m m β‐NAD + for 30 min at 37 °C.

Techniques: Recombinant, Incubation, Purification, SDS Page, Autoradiography, Mutagenesis

Journal: The Febs Journal

Article Title: ENPP 1 processes protein ADP ‐ribosylation in vitro

doi: 10.1111/febs.13811

Figure Lengend Snippet: ENPP 1 hydrolyses poly( ADP ‐ribose) to pR, a molecular tag detectable by LC ‐ MS / MS . (A) PARP 1 carries a 212.01 Da shift representative of pR on E168. (B) PARP 1 carries pR on E169, as shown here clearly distinguishable from the pR‐E168 peptide form. Both peptides detected following digestion of PAR by ENPP 2‐1‐T in a 10× enzyme:PARP ratio.

Article Snippet: The 6xHis‐ hs PARP1 (wild‐type) was expressed and purified from E. coli , attached to MagneHis beads (Promega Corporation, Madison, WI, USA), and PARylated as described previously with the following changes: PARP1 (final concentration 1 μ m ) was autoPARylated in the presence of 1 m m β‐NAD + for 30 min at 37 °C.

Techniques: Liquid Chromatography with Mass Spectroscopy

Journal: PLoS ONE

Article Title: M30 Antagonizes Indoleamine 2,3-Dioxygenase Activation and Neurodegeneration Induced by Corticosterone in the Hippocampus

doi: 10.1371/journal.pone.0166966

Figure Lengend Snippet: Levels of protein expression of (A) Bcl-2, (B) Cleaved Caspase 3 and (C) Cleaved PARP-1 in the hippocampus of the control, CORT-treated (CORT), M30-treated (M30), CORT and M30 co-treated (CORT+M30) or vehicle groups are summarized. β-actin was an internal control. Data from each group were expressed as mean ± SEM (n = 8). Statistical comparisons between groups were performed using the One way Anova followed by Tukey post hoc test to detect differences in all groups. For Bcl-2, *p < 0.01 when compared with Control, # p < 0.01 when compared with M30, $ p < 0.01 when compared with CORT + M30 groups, ! p < 0.01 when compared with Vehicle. For cleaved caspase 3 and cleaved PARP-1, *p < 0.001 when compared with Control, # p < 0.001 when compared with M30, $ p < 0.001 when compared with CORT + M30 groups, ! p < 0.001 when compared with Vehicle.

Article Snippet: Primary antibodies of SOD-2 (rabbit polyclonal, 1:1000), GPx-1 (goat polyclonal, 1:500), NFκB p65 (rabbit polyclonal, 1:250) and p50 (mouse monoclonal, 1:250), IκBα (mouse monoclonal, 1:500), TNF α (goat polyclonal, 1:80), IL-1β (rabbit polyclonal 1:100), IL-6 (goat polyclonal, 1:1000) and COX-2 (goat polyclonal, 1:100) were purchased from Santa Cruz Biotechnology, CA, USA; Synapsin 1 (rabbit polyclonal, 1:500) and Synaptophysin (rabbit polyclonal, 1:2000) were purchased from Novus Biologicals, USA; PSD95 (rabbit polyclonal 1:500), Cleaved Caspase 3 (rabbit polyclonal 1:500) was purchased from Cell Signaling Technology; Cleaved PARP-1 (rabbit polyclonal, 1:2000) was purchased from Bioworld Technology; IDO-1 (rabbit polyclonal, 1:250) was purchased from antibodies-online (ABIN1714836).

Techniques: Expressing