ethylene glycol tetraacetic acid egta  (Millipore)

Journal: The Journal of Cell Biology

Article Title: Herpes simplex virus triggers activation of calcium-signaling pathways

doi: 10.1083/jcb.200301084

Figure Lengend Snippet: 2-APB and BAPTA-AM prevent viral-induced FAK phosphorylation. CaSki cells were serum starved overnight, and a synchronized infection with HSV-2(G) was conducted as described in Materials and methods. 100 μM 2-APB, 10 μM nifedipine, or control buffer (5% DMSO) was added to cells at the time of binding or penetration (temperature shift, 15 min, 37°C). Cells were treated with low pH citrate buffer, washed, and cell lysates were prepared 10 min after citrate treatment. Western blots were prepared, probed with anti-FAK pY 397 , and scanned by optical densitometry. Blots were then stripped and reprobed with mAb to total FAK. A representative blot is shown in A. Alternatively, cells were pretreated for 2 h with BAPTA-AM, EGTA, or 5% methanol in PBS (control), and then exposed to HSV-2(G). (B) Lysates were prepared 5 min after infection and analyzed by Western blotting for FAK phosphorylation. (C) Results obtained from three independent experiments are summarized graphically as odu of phosphorylated FAK:odu total FAK as a percentage of control (HSV-2(G) in the absence of any inhibitors). Results are the mean ± SD obtained from three independent experiments; asterisks indicate P

Article Snippet: Reagents Verapamil, nifedipine, 2-APB, Tg, EGTA, EGTA-AM, and ionomycin were purchased from Calbiochem and diluted in DMSO or PBS per manufacturer's instructions.

Techniques: Infection, Binding Assay, Western Blot

Journal: The Journal of Cell Biology

Article Title: Herpes simplex virus triggers activation of calcium-signaling pathways

doi: 10.1083/jcb.200301084

Figure Lengend Snippet: The ER Ca 2+ release is required for HSV infection. The effects of pretreating cells for 1 h with 50 μM BAPTA-AM, 0.5 mM EGTA, or adding 100 μM 2-APB, 10 μM nifedipine, or 10 μM verapamil during viral penetration on HSV-1(KOS), HSV-2(G), or VSV infection of Vero cells (top) or CaSki cells (bottom) were examined. Results are presented as pfu formed in the presence of the indicated concentration of drug as a percentage of pfu formed in cells treated with control buffer (5% DMSO for pharmacological inhibitors or 5% methanol for BAPTA), and are means of at least three independent experiments conducted in duplicate. Cytotoxicity of pharmacological agents was determined in parallel after a 2-h exposure to drug and quantifying viable, proliferating cells at 24 h by MTS assay. Cell viability results are expressed as odu reading in the presence of the drug as a percentage of odu reading in the presence of control buffer, and are means of two independent experiments conducted in triplicate. Asterisks denote P

Article Snippet: Reagents Verapamil, nifedipine, 2-APB, Tg, EGTA, EGTA-AM, and ionomycin were purchased from Calbiochem and diluted in DMSO or PBS per manufacturer's instructions.

Techniques: Infection, Concentration Assay, MTS Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Functional cleavage of the common cytokine receptor ? chain (?c) by calpain

doi:

Figure Lengend Snippet: Cleavage of γ c by calpain. In vitro -translated wild-type human γ c ( A ) and γ c in which the PEST sequence was mutated ( B ) were treated with m-calpain and run on 4–20% SDS gels. Although mature γ c is approximately 64 kDa, in vitro -translated γ c migrates at approximately 42 kDa, at least in part due to the lack of glycosylation. We confirmed a report that in vitro ). Murine wild-type and PEST-mutated γ c yielded similar results to those shown for human γ c (data not shown). ( C ) Proteolysis of γ c by calpain in YT cells. YT cell lysates were incubated with 5 mM CaCl 2 (lanes 4, 5, and 7–10) or 10 mM EGTA + 2.5 mM EDTA (lanes 3 and 6) at 37°C for 0–60 min, and reactions were stopped by the addition of 10 mM EGTA/10 μg/ml leupeptin (Boehringer Mannheim)/240 μg/ml 4-[2-aminoethyl]-benzenesulfonyl fluoride hydrochloride/10 μg/ml aprotinin (ICN). In lanes 4, 7, and 10, 20 μM calpastatin or 50 μg/ml antipain also were added. Reactions were stopped with 10 mM EGTA and the protease inhibitor mix. Lysates were immunoprecipitated with chicken anti-human γ c (lanes 2–10) or control chicken IgY (lane 1), and immunoblotted using R878 antiserum to γ c . As R878 antiserum recognizes the C-terminal end of γ c , cleavage in the cytoplasmic domain would result in immunoreactive fragments too small to be retained on these gels. The cleavage was specific, as shown by the lack of general degradation of cellular proteins as detected by Coomassie staining (data not shown).

Article Snippet: Lysates were centrifuged at 15,000 rpm for 20 min at 4°C and incubated with CaCl2 (final concentration 5 mM) or EGTA (final concentration 10 mM) at 37°C for 0 to 60 min. To determine the specificity of proteolysis by calpain, calpastatin (20 μM, Calbiochem), an endogenous protease inhibitor that acts specifically on calpain, or antipain [S-(1 carboxy-2-phenylethyl)- l -carbamyl= l -arginyl- l -valylargininal] (Calbiochem), another potent inhibitor of calpain, were incubated in the presence of 5 mM CaCl2 .

Techniques: In Vitro, Sequencing, Incubation, Protease Inhibitor, Immunoprecipitation, Staining

Journal: PLoS ONE

Article Title: Activation of AMPK by Bitter Melon Triterpenoids Involves CaMKK?

doi: 10.1371/journal.pone.0062309

Figure Lengend Snippet: BMTs activate AMPK through CaMKK and not through intracellular Ca 2+ changes. Serum-starved L6 myotubes were treated with either vehicle (V) alone, BMT-17 at 0.1 µM, 1 µM or 10 µM for 30 min before lysis; 10 µg of lysate was then analysed by Western blot ( A ) and quantified by densitometry ( B ). A representative blot is shown. Serum-starved L6 myotubes were pre-treated with either 150 µM EGTA-AM (+) or diluent (-) for 15 min before then being treated with 10 µM BMT-17 or vehicle for a further 30 min before lysis. AMPK was then isolated from 50 µg lysate by pan-AMPKβ immunoprecipitation and assessed by AMPK in vitro kinase assay ( C ). Data are means relative to untreated vehicle control (V) ± SEM from 5 independent experiments. *p

Article Snippet: The inability of EGTA-AM to block the BMT-dependent AMPK activation demonstrated that the BMT-dependent AMPK activation was independent of intracellular calcium levels.

Techniques: Lysis, Western Blot, Isolation, Immunoprecipitation, In Vitro, Kinase Assay

Journal: Journal of Neurochemistry

Article Title: Synaptic vesicle exocytosis and increased cytosolic calcium are both necessary but not sufficient for activity‐dependent bulk endocytosis

doi: 10.1111/jnc.13132

Figure Lengend Snippet: Dynamin I dephosphorylation is arrested by BAPTA ‐ AM and EGTA ‐ AM . Cultures were preincubated with BAPTA ‐ AM , EGTA ‐ AM (both 100 μM) or DMSO (vehicle control) for 30 min. They were then either stimulated with a train of 800 action potentials (80 Hz) or left to rest for 10 s. Cultures were then immediately lysed in SDS sample buffer. (a) Representative immunoblots of cerebellar granule neuron lysates probed with either dynamin I phospho‐Ser774 ( PD ynI) or synaptophysin (Syp) antibodies. (b) Quantification of PD ynI levels after normalisation against Syp ( PD ynI/Syp) ± SEM . Blue bars represent Ctrl, red bars BAPTA ‐ AM and purple bars EGTA ‐ AM , *** p

Article Snippet: The inhibition of dynamin I dephosphorylation in the presence of EGTA‐AM is consistent with this event being central in the triggering of this endocytosis mode.

Techniques: De-Phosphorylation Assay, Western Blot