Journal: Science signaling

Article Title: Fluorescent Ca 2+ indicators directly inhibit the Na,K-ATPase and disrupt cellular functions

doi: 10.1126/scisignal.aal2039

Figure Lengend Snippet: (a) Representative images of astrocytes loaded with Rhod-2 AM (4.5 μM) or prepared from GFAPCre:GCaMP3fl transgenic mice (GFAP-GCaMP3). Scale bar, 100 μm. (b) GCaMP3 expression in astrocytes has no effect on Na,K-ATPase-mediated 86Rb+ uptake. GFAP-GCaMP3 astrocytes showed no difference in ouabain-sensitive 86Rb+ uptake. Expression of GCaMP3 induced by adenovirus bearing Cre recombinase (100 MOI, 6 days post-transduction) in cultured astrocytes prepared from GCaMP3fl transgenic mice also had no effect on ouabain-sensitive 86Rb+ uptake (AdV-GCaMP3). Conversely, Rhod-2 AM (4 μM) loading decreased ouabain-sensitive 86Rb+ uptake in all groups of cultured mouse astrocytes. n=6–23 wells; *p<0.05, **p<0.01, ****p<0.0001 compared to (−) Rhod-2 AM of control group (0.1% DMSO). (c) Spontaneous Ca2+ activity in cultured mouse astrocytes. Number of spontaneous Ca2+ events were detected by Fluo-4 AM (2 and 4 μM), Rhod-2 AM (2.25 and 4.5 μM) or GCaMP3. In groups combining Ca2+ imaging of GCaMP3 with Rhod-2 AM, number of Ca2+ events detected with Rhod-2 (left, orange channel) and GCaMP3 (right, green channel) were both counted. n=4–6 wells; ****p<0.0001 compared to GFAP-GCaMP3 control group (0.1% DMSO). (d) Histogram comparing number of spontaneous Ca2+ events in Adv-GCaMP3 expressing mouse astrocytes in the presence and absence of 5,5’-Dibromo BAPTA (p=0.4188, unpaired t test n=4–8 wells). Displayed are means ± S.E.M.

Article Snippet: Titration of adenoviral infection of GCaMP3fl astrocytes To induce GCaMP3 expression in cortical mouse astrocyte cultures prepared from GCaMP3fl mice, cells were infected with adenovirus bearing Cre recombinase under the control of the constitutive cytomegalovirus (CMV) promoter (courtesy of Aris N. Economides from Regeneron Pharmaceuticals, Tarrytown, NY) for 5 h with 10, 50 or 100 multiplicities of infection (MOI).

Techniques: Transgenic Assay, Expressing, Transduction, Cell Culture, Control, Activity Assay, Imaging

Journal: Methods in enzymology

Article Title: Effective Isotope Labeling of Proteins in a Mammalian Expression System

doi: 10.1016/bs.mie.2015.09.021

Figure Lengend Snippet: Recombinant adenovirus as a tool to obtain isotopically labeled proteins. The target gene is cloned into a shuttle vector (pVRC1290) using the restriction sites (e.g., Xba1, BamH1) in the multiple cloning site; adenoviral cosmid DNA (pVRC1194) and shuttle vector are each linearized with ClaI and PacI, respectively, then recombined in vitro with Cre–Lox recombinase to obtain recombinant adenoviral genome (Aoki et al., 1999). The recombined adenoviral type 5 DNA is transfected into 293 adherent helper mammalian cells and recombinant adenovirus is isolated and purified using well-established methods. Target protein production is achieved by infecting CAR+ mammalian cells, such as A549 or CHO(CAR+).

Article Snippet: Equimolar amounts of the shuttle plasmid and the adenoviral cosmid are recombined in vitro using Cre recombinase (New England Biolabs), recombination reaction is monitored using gel electrophoresis and upon completion, the recombinant adenoviral genome is purified by standard phenol/chloroform extraction and ethanol precipitation.

Techniques: Recombinant, Labeling, Clone Assay, Plasmid Preparation, Cosmid DNA, In Vitro, Transfection, Isolation, Purification