phenylmethylsulfonyl fluoride  (Millipore)

Journal: Molecular Metabolism

Article Title: Tannic acid, a novel histone acetyltransferase inhibitor, prevents non-alcoholic fatty liver disease both in vivo and in vitro model

doi: 10.1016/j.molmet.2018.11.001

Figure Lengend Snippet: Inhibitory effects of TA on HAT activity. (A) A colorimetric assay was performed to measure HAT activity in the presence of TA at the indicated concentrations. The results are presented as percentages relative to the control sample, which was incubated without TA (left panel). The IC 50 value of TA inhibition of HAT activity was calculated using Prism software (right panel). (B) An in vitro HAT assay using autoradiography was performed to evaluate the effect of the anti-HAT activity of TA on the acetylation of a synthetic H4 peptide by a HeLa NE. The arrow indicates histone H4 (left panel). The autoradiography was quantified using Image J software (right panel). The values presented are the means ± SD of two independent experiments. Means with different superscript letters are significantly different, p

Article Snippet: For autoradiography-based in vitro HAT activity assays, HeLa cell nuclear extract was incubated with HAT assay buffer (50 mM HEPES, pH 8.0; 10% glycerol; 1 mM DTT; 1 mM phenylmethylsulfonyl fluoride; and 10 mM sodium butyrate), 1 μL [3 H] acetyl-CoA, and 5 μg of biotinylated-H4 peptide (Millipore, Billerica, MA, USA) along with TA at the indicated concentrations at 30 °C for 1 h. The reactions were stopped by adding 5 × sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) sample buffer and samples were separated on 15% SDS–PAGE gels, followed by autoradiographic analysis.

Techniques: HAT Assay, Activity Assay, Colorimetric Assay, Incubation, Inhibition, Software, In Vitro, Autoradiography

Journal: Redox Biology

Article Title: Matrix metalloproteinase-2-induced epidermal growth factor receptor transactivation impairs redox balance in vascular smooth muscle cells and facilitates vascular contraction

doi: 10.1016/j.redox.2018.07.005

Figure Lengend Snippet: MMP inhibitors prevent the increases in vascular gelatinolytic activity, in ROS concentrations, and in lucigenin chemiluminescence after intraluminal incubation of rabbit aorta with MMP-2. (A) Representative photomicrographs of aortic in situ zymography experiments showing that MMP-2 gelatinolytic activity was totally inhibited by phenanthroline (Phe, 100 µM) or doxycycline (Doxy, 100 µM), but not by phenylmethylsulfonyl fluoride (PMSF, 100 µM). The arteries were incubated with the inhibitors together with MMP-2 (16 nM) intraluminally for 30 min. (B) Quantification of aortic gelatinolytic activity measured in experiments described in A. (C) Representative photomicrographs of aortic sections incubated with DHE. Red fluorescence reflects ROS production and shows that incubation with MMP-2 (16 nM) intraluminally for 30 min increased ROS production, and this effect was totally inhibited by phenanthroline (Phe, 100 µM) or doxycycline (Doxy, 100 µM), but not by phenylmethylsulfonyl fluoride (PMSF, 100 µM). The experiments were carried out as described in A. (D), Quantification of aortic fluorescence of DHE measured in experiments described in C. (E), Quantification of lucigenin chemiluminescence measured in fresh aortas after intraluminal incubation with MMP-2 (16 nM) or vehicle for 30 min in absence or presence of MMP inhibitors, doxycycline (Doxy 100 µM) or GM6001 (10 µM). Data are shown as the mean ± SEM (n = 4–5/group). * P

Article Snippet: Tyrphostin AG 1478, Phenylephrine, Apocynin, Peg-Catalase (PG-Cat), Dihydroethidium (DHE), phenanthroline, Phenylmethylsulfonyl fluoride were purchased from Sigma Chemical Co. (St. Louis, MO, USA).

Techniques: Activity Assay, Incubation, In Situ, Zymography, Fluorescence

Journal: Molecular and Cellular Biology

Article Title: A Novel Pathway for Mammary Epithelial Cell Invasion Induced by the Helix-Loop-Helix Protein Id-1

doi:

Figure Lengend Snippet: Expression of a 120-kDa gelatinase by Id-1-expressing mammary epithelial cells. (A) Gelatin zymogram of conditioned media from SCp2–Id-1A (lane 1) and SCp2–Id-1E (lane 2) cells. Cells were cultured in serum-free medium, and conditioned media were harvested and analyzed on a gelatin substrate gel, as described in Materials and Methods. (B) Gelatin zymogram of conditioned media from SCp2–Id-1A cells either growth arrested by serum deprivation (lane 1) or growing in 5% serum (lane 2). (C) Gelatin zymogram of conditioned media from control SCp2 cells (lane 1) and an uncloned SCp2–Id-1-transfected pooled population (lane 2). Cells were cultured in serum-free medium. (D) Gelatin zymogram of SCp2–Id-1A (lanes 1, 3, and 5) and SCp2–Id-1E (lanes 2, 4, and 6) cell-conditioned media incubated with DMSO (lanes 1 and 2), the MMP inhibitor GM6001 (0.2 mM in DMSO) (lanes 3 and 4), or the serine proteinase inhibitor PMSF (5 mM in DMSO) (lanes 5 and 6). (E) Casein zymogram of conditioned media from SCp2–Id-1A (lanes 1 and 3) and SCp2–Id-1E (lanes 2 and 4) cells incubated with DMSO (lanes 1 and 2) or GM6001 (lanes 3 and 4). In panels A through D, arrows mark the positions of the 120-kDa MMP.

Article Snippet: Concentrated conditioned medium was mixed with nonreducing Laemmli sample buffer and incubated at 37°C for 15 min. After electrophoresis, the gels were incubated for 1 h in 2.5% Triton X-100 at room temperature, followed by 24 to 48 h in substrate buffer (100 mM Tris-HCl [pH 7.4]–15 mM CaCl2 ) in the absence or presence of GM6001 (0.2 mM in DMSO; supplied by Glycomed Corporation and obtained from Z. Werb [ ]), EDTA (10 mM), ortho -phenanthroline (1 mM in DMSO; Sigma), PMSF (5 mM), or AEBSF (0.5 mM; Calbiochem).

Techniques: Expressing, Cell Culture, Transfection, Incubation

Journal: Cell motility and the cytoskeleton

Article Title: The Actin-binding Domain of Cortactin is Dynamic and Unstructured and Affects Lateral and Longitudinal Contacts in F-actin

doi: 10.1002/cm.20328

Figure Lengend Snippet: The cortactin repeat has a dynamic conformation in solution (A) The endogenous cysteine residues 112 and 246 of the cortactin repeat were crosslinked and the ability of the crosslinked protein to bind F-actin was tested in a co-sedimentation assay (Materials and Methods). The pellet (P) and supernatant (S) were analyzed on 12% SDS-PAGE. Lanes 1 and 2, uncrosslinked cortactin; lanes 3 and 4, disulfide crosslinked cortactin (xl-cortactin); lanes 5 and 6, reduction of the disulfide crosslink with 10mM DTT. (B) The ability of the NEM-labeled cortactin repeat (at residues C112 and C246) to bind to F-actin was assessed using a co-sedimentation assay (Materials and Methods). The pellet and supernatant were analyzed on 12% SDS-PAGE. Lanes 1 and 2, non-labeled cortactin; lanes 3 and 4, NEM-labeled cortactin. (C) Crosslinking of cortactin repeat constructs containing pairs of cysteine residues at positions 83 and 306 (left), 83 and 246 (middle), and 83 and 112 (right). Prior to each crosslinking reaction, DTT was removed from the samples on a Sephadex G-50 spin column equilibrated with 20 mM HEPES pH 7.0, 0.1 M KCl, and 100μM PMSF. The crosslinking reactions were performed on ice and catalyzed by addition of 10μM CuSO 4 . Each cysteine pair was crosslinked either directly by disulfide bond formation (zero length crosslink), or using the crosslinking reagents DBB (4.4 Å span) or MTS-6 (9.6 Å span).

Article Snippet: Sephadex G-50, N-ethylmaleimide (NEM), ATP, phalloidin, DTT and PMSF were purchased from Sigma Chemical Company (St. Louis, MO).

Techniques: Sedimentation, SDS Page, Labeling, Construct