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Methods in molecular exercise physiology
Published in Adam P. Sharples, James P. Morton, Henning Wackerhage, Molecular Exercise Physiology, 2022
Adam P. Sharples, Daniel C. Turner, Stephen Roth, Robert A. Seaborne, Brendan Egan, Mark Viggars, Jonathan C. Jarvis, Daniel J. Owens, Jatin G. Burniston, Piotr P. Gorski, Claire E. Stewart
The extraction of protein from tissue samples was briefly introduced earlier in this chapter. In the case of Western blotting of skeletal muscle samples, the ordered structure of myofibrils and connective tissue means that skeletal muscle is relatively difficult to prepare for protein assays compared to cell culture lysates. Grinding the muscle samples under liquid nitrogen using a mortar and pestle (or similar approach), and then processing the debris/powder in a relevant homogenisation/lysis buffer with an electric tissue homogeniser have the effect of shearing the cells and organelles and breaking up the connective tissue. A typical homogenisation buffer for extracting soluble proteins is a pH-buffered solution (pH 7–9) containing non-ionic detergents such as Triton x-100 to disrupt membranes and protein interactions, reducing agents such as dithiothreitol (DTT) to break disulphide bonds, and other agents such as EDTA and EGTA to inhibit protein-degrading proteases. When post-translational modifications are of interest, other agents such as phosphatase inhibitors (e.g. sodium orthovanadate and sodium fluoride) or deacetylase inhibitors (e.g. trichostatin A and nicotinamide) are included to avoid dephosphorylation and acetylation, respectively, during protein extraction. Inhibitor ‘cocktails’ that can be added to standard homogenisation buffers are now commercially available and are formulated specifically for preserving the various post-translational modifications of interest.
Endothelial Cell Signaling During Wound Healing
Published in John J. Lemasters, Constance Oliver, Cell Biology of Trauma, 2020
FAK activation was examined by assaying autophosphorylation67 using previously described techniques.42 FAK immunoprecipitates were obtained from cell lysates and bound to protein A sepharose as described above. These complexes were washed with lysis buffer and then with TBS prior to transfer to 3 mM MnCl2 with 10 mM PIPES, pH 7.46. Then 10 μCi of 32P-labeled γATP (New England Nuclear, Wilmington, DE) were incubated with each sample at room temperature for 20 min. Laemmli sample buffer containing 1 mM sodium orthovanadate was then added, and samples were boiled for 3 min. The 32P-labeled FAK was then analyzed with SDS-PAGE along with molecular weight marker proteins; 10% polyacrylamide gels were used with bisacrylamide added at a concentration of 0.13%. These gels were then stained with Coomassie blue and dried. Autoradiography was accomplished by exposure to X-ray film and intensifying screens at -70°C.
Drug Targeting to the Lung: Chemical and Biochemical Considerations
Published in Anthony J. Hickey, Sandro R.P. da Rocha, Pharmaceutical Inhalation Aerosol Technology, 2019
Peter A. Crooks, Narsimha R. Penthala, Abeer M. Al-Ghananeem
In a structure-activity correlation study, a number of N-substituted derivatives of rolipram (52) were prepared and evaluated (Tanaka et al. 1995). A carbamate ester of rolipram was found to be approximately 10-fold more potent than rolipram itself at inhibiting human PDE IV. A methyl ketone derivative of rolipram showed more potent inhibition of PDE IV compared to rolipram or its carbamate ester. Based on proton NMR spectroscopy and computer modeling studies, a pharmacophore model of the methyl ketone derivative was proposed (Stafford et al. 1995). This model showed that the ketone carbonyl oxygen atom is involved in an important interaction within the PDE IV active site. Sodium orthovanadate, a phosphotyrosine phosphate inhibitor, exhibits dose- and time-dependent suppression of Lewis lung carcinoma A11 cell spreading. Protein tyrosine phosphorylation levels in A11 cells were elevated after treatment with ortho vanadate; this increase was partially diminished by the tyrosine kinase inhibitor ST 638, concomitantly with restoration of the suppressed cell spreading, as well as invasive and metastatic ability (Takenaga 1996). These results suggest tyrosine phosphorylation influences adhesion of cancer cells to lung surface endothelia, and that a valid approach in treating cancer is inhibition of phosphotyrosine phosphatase.
Sauropus brevipes ethanol extract negatively regulates inflammatory responses in vivo and in vitro by targeting Src, Syk and IRAK1
Published in Pharmaceutical Biology, 2021
Ji Hye Kim, Jae Gwang Park, Yo Han Hong, Kon Kuk Shin, Jin Kyeong Kim, Young-Dong Kim, Ki Dong Yoon, Kyung-Hee Kim, Byong Chul Yoo, Gi-Ho Sung, Jae Youl Cho
To obtain total cell lysate, harvested cells were first washed with cold PBS containing 1 mM sodium orthovanadate. The washed cells were then lysed in ice-cold modified RIPA buffer (50 mM Tris–HCl, pH 7.4, 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM Na3VO4 and 1 mM NaF) including protease inhibitors (2 mM PMSF, 100 μg/mL leupeptin, 10 μg/mL pepstatin, 1 μg/mL aprotinin and 2 mM EDTA) for 30 min with rotation at 4 °C. At this time, a sonicator (Thermo Fisher Scientific, Waltham, MA) was used to improve cell lysis efficiency as reported previously (Choi et al. 2019). Lysates were refined by centrifugation at 16,000×g for 10 min at 4 °C and stored at −20 °C until use. To prepare nuclear lysate, a three-step procedure was performed. In the first step, harvested cells were washed and lysed with 500 mL lysis buffer (50 mM KCl, 0.5% Nonidet P-40, 25 mM HEPES, 1 mM phenylmethylsulfonyl fluoride, 10 μg/mL leupeptin, 20 μg/mL aprotinin and 100 μM 1,4-dithiothreitol) on ice for 4 min. Second, lysates were centrifuged at 16,000×g for 1 min. The pellet obtained from lysates was washed with washing buffer (lysis buffer without Nonidet P-40). Finally, the pellet containing nuclei was lysed with an extraction buffer (lysis buffer with 500 mM KCl and 10% glycerol). The nuclei/extraction buffer mixture was frozen at −80 °C, and then centrifuged at 16,000×g for 5 min. The supernatant was collected as a nuclear extract.
Inhibition of EphA/Ephrin-A signaling using genetic and pharmacologic approaches improves recovery following traumatic brain injury in mice
Published in Brain Injury, 2019
Shavonne Teng, Alicia Palmieri, Isabella Maita, Cynthia Zheng, Gitanjali Das, Juyeon Park, Renping Zhou, Janet Alder, Smita Thakker-Varia
Sodium orthovanadate has not been previously examined in TBI, but studies have indicated neuroprotective effects of this PTP2 inhibitor in various brain diseases and injuries such as vascular dementia and hypoxic-ischemic injury (65,68,69). However, due to the broad effects of sodium orthovanadate (70,71), it is possible that this compound may exert other effects which are not specific for Eph signaling. Future studies would confirm the effectiveness of sodium orthovanadate in other models of injury and species. Further evaluation on sodium orthovanadate as an approach for improving outcomes after TBI can be considered as well as exploration of other EphA6 signaling pathways in the cellular and behavioral recovery from TBI.