pires egfp expression vector  (TaKaRa)

Journal: eLife

Article Title: Enhanced functional detection of synaptic calcium-permeable AMPA receptors using intracellular NASPM

doi: 10.7554/eLife.66765

Figure Lengend Snippet:

Article Snippet: Transfected construct ( Homo sapiens ) , pIRES-eGFP-γ7 , doi: 10.1038/nn.2266 , , CACNG7 ; OriGene Technologies pCMV6-XL4-γ7 (subcloned into pIRES-eGFP).

Techniques: Mutagenesis, Transfection, Construct, Software

Journal: The Journal of Experimental Medicine

Article Title: IL-24 intrinsically regulates Th17 cell pathogenicity in mice

doi: 10.1084/jem.20212443

Figure Lengend Snippet: Intracellular IL-24 localizes to the inner mitochondrial membrane and is necessary and sufficient for IL-10 production by Th17 cells. (a) Naive T cells from Il24 +/+ wild-type or Il24 −/− mice were cultured in Th17 differentiation conditions for 3 d. Th17 cells were nucleofected with pIRES-eGFP, pIRES-eGFP-IL-24, or pIRES-eGFP-IL-24t on day 1 and analyzed for intracellular IL-10 on day 3 with representative FACS plots (left, pregated for viable CD4 + GFP + cells) and summary of four independent experiments (mean ± SEM) normalized to wild type (right). Relative fraction indicates IL-10 + frequency (among CD4 + cells) for the respective nucleofection, divided by the corresponding IL-10 + frequency of the Il24 +/+ pIRES-eGFP nucleofected (control) cells for each experiment. Asterisks indicate significance level of Dunnett’s multiple comparison test after one-way ANOVA (*, P < 0.05). (b) Naive T cells from wild-type mice were cultured in Th17 differentiation conditions. On day 2 of differentiation, cells were nucleofected with mRuby2-N1-IL-24t (top row) or mRuby2-N1-IFN-γt (bottom row) and analyzed for intracellular cytokine distribution by confocal microscopy. Mitochondria were marked with Mitotracker. Representative microphotographs; scale bar, 2 μm (left). Quantification of the cytokine signal in the mitochondrial compartment (right) from three independent experiments (mean ± SEM). Asterisk indicates statistical significance of a two-tailed t test. (c) Naive wild-type or Il24 −/− T cells mice were differentiated into Th17 cells. On day 3, mitochondrial (Mito.) and nuclear (Nucl.) fractions were isolated and probed for STAT3. Lamin B1 and NDUFA9 were used as loading controls for nuclear and mitochondrial fractions, respectively (left). Densitometric quantification (right) of three independent experiments (mean ± SEM). Asterisk indicates statistical significance of a two-tailed t test. (d) Grim19 interacts with STAT3 in an IL-24–dependent manner. Naive T cells from wild-type or Il24 −/− mice were differentiated into Th17 cells. On day 3, cells were transferred into medium containing IL-2 (2 ng/ml). After 24 h, mitochondrial fractions were isolated, solubilized, immunoprecipitated with anti-Grim19 monoclonal antibody (Grim19 IP ), and probed for the amount of pulled STAT3 by Western blot. Solubilized mitochondrial fraction (input) was used as a control for coimmunoprecipitation. Representative Western blots from three independent experiments. (e) Loss of STAT3 from nuclear compartment. Naive T cells from wild-type or Il24 −/− mice were cultured in Th17 differentiation conditions for 3 d. After 3 d, cells were transferred into IL-2 medium (2 ng/ml) and analyzed for STAT3 in the nuclear compartment at baseline and after 6, 12, and 24 h. Representative Western blots (left) with normalized densitometry data (right) quantified from three to six independent experiments (mean ± SEM). Asterisks indicate significance level of Sidak’s multiple comparison test following two-way ANOVA (*, P < 0.05). Source data are available for this figure: .

Article Snippet: The sequences corresponding to full-length or nonsecretable (truncated) versions of IL-24, i.e., IL-24 and IL-24t, were cloned into mammalian expression vector pIRES-eGFP (6029-1; Clontech).

Techniques: Cell Culture, Confocal Microscopy, Two Tailed Test, Isolation, Immunoprecipitation, Western Blot

Journal: Cell Death Discovery

Article Title: Loss of function of Ywhah in mice induces deafness and cochlear outer hair cells' degeneration

doi: 10.1038/cddiscovery.2016.17

Figure Lengend Snippet: Impact of YWHAH variants in human fibroblasts. Fibroblasts from patients (family 1: the father ∆1 and his son ∆2, family 2: D236N) were compared in all experiments with three control fibroblast cultures obtained from healthy patients. ( a ) Susceptibility to apoptosis was assessed by counting apoptotic nuclei after a 3-h staurosporine treatment (1 μ M; n =3). ( b ) Mitochondrial network structure was assessed on exponential growing cultures after Mitotracker staining. Filamentous, fragmented and punctuated phenotypes were counted on 200 cells in 3 independent experiments. Data are mean±S.E.M. ( c ) HeLa cells were transiently transfected with pIRES-EGFP vectors expressing 14-3-3eta, 14-3-3eta ∆189 and 14-3-3eta D236N. Western blotting of total cell lysate showing the expression of the different proteins (arrows). Actin was used as a loading control. ( d ) In a parallel experiment, HeLa cells were treated with 1 μ M staurosporine for 3 h before the quantification of apoptotic cells. Apoptosis was assessed by immunofluorescent detection (top) of the active form of caspase - 3 (red fluorescence) in transfected cells (green fluorescence). All cells were stained with the nuclear marker Hoechst. Scale bar represents 20 μ m. Quantification of apoptosis in transfected cells (bottom), each point representing the average of triplicate±S.E.M.

Article Snippet: Mammalian expression vectors for 14-3-3eta, 14-3-3eta Δ189 and 14-3-3eta D236N were constructed by PCR amplification of the corresponding human cDNA fragment and subsequently cloned into CMV promoter-based expression vectors pIRES-EGFP (Clontech, Mountain View, CA, USA) or pcDNA/V5-His vector (Invitrogen).

Techniques: Staining, Transfection, Expressing, Western Blot, Fluorescence, Marker