shrna sk1  (OriGene)

Journal: Biochimica et biophysica acta

Article Title: A novel chimeric aequorin fused with caveolin-1 reveals a sphingosine kinase 1-regulated Ca²⁺ microdomain in the caveolar compartment.

doi: 10.1016/j.bbamcr.2015.04.005

Figure Lengend Snippet: Fig. 3. The effect of SK1-overexpression on [Ca2+] at the plasma membrane as reported by SNAP25–aequorin (SNAP–Aeq) and in the cytosol as reported by Fura-2 AM. (A) Representative traces showing [Ca2+]PM during histamine (100 μM) stimulation in presence of EGTA (150 μM), and Ca2+ (1 mM) re-addition (ROCE). Bar diagrams show the mean with S.E.M. indicating the change in [Ca2+]PM during histamine-stimulation (B), and during Ca2+ re-addition (C), n = 5. (D) Average traces showing the Fura-2 AM fluorescence ratio (F340/F380) upon the same experimental conditions as in (A). The bar di- agrams show the mean with S.E.M. indicating the change in the fluorescence ratio (ΔF340/ F380) during histamine stimulation in the presence of EGTA (E) and during Ca2+ re-addi- tion (F), n = 6, *P b 0.05.

Article Snippet: The lentiviral particles for SK1 shRNA (sc156038-V), as well as the control vectors (sc-108080), were acquired from Santa Cruz Biotechnology and the cells were transduced by incubating the cells for 12 h with polybrene (8 mg/ml) along with the lentiviral particles at multiplicity of infection 30.

Techniques: Over Expression, Clinical Proteomics, Membrane

Journal: Biochimica et biophysica acta

Article Title: A novel chimeric aequorin fused with caveolin-1 reveals a sphingosine kinase 1-regulated Ca²⁺ microdomain in the caveolar compartment.

doi: 10.1016/j.bbamcr.2015.04.005

Figure Lengend Snippet: Fig. 4. Overexpression of SK1 induces an increase in [Ca2+]cav during histamine-stimulated release of endoplasmic Ca2+, as well as during receptor-operated Ca2+ entry (ROCE). The cells were pretreated for 1 h with either 1 μM VPC and 10 μM JTE in combination, or with the control solutions. Following the pre-incubation, the compounds were present throughout the experiments. A) Representative traces of experiments showing [Ca2+]cav during histamine (100 μM) stimulation in presence of EGTA (150 μM), and Ca2+ (1 mM) re-addition (ROCE). B) Quantification of the change in [Ca2+] at the caveolae during histamine-induced Ca2+ mobilization in the absence of extracellular Ca2+. C) Quantification of the change in [Ca2+]cav during Ca2+ re-addition in the presence of histamine. The bar diagrams show the mean with S.E.M., n = 7–10, *P b 0.05, ***P b 0.001 (mock control vs hSK1 control), ¤¤ ¤P b 0.001 (hSK1 control vs hSK1 VPC + JTE).

Article Snippet: The lentiviral particles for SK1 shRNA (sc156038-V), as well as the control vectors (sc-108080), were acquired from Santa Cruz Biotechnology and the cells were transduced by incubating the cells for 12 h with polybrene (8 mg/ml) along with the lentiviral particles at multiplicity of infection 30.

Techniques: Over Expression, Control, Incubation

Journal: Biochimica et biophysica acta

Article Title: A novel chimeric aequorin fused with caveolin-1 reveals a sphingosine kinase 1-regulated Ca²⁺ microdomain in the caveolar compartment.

doi: 10.1016/j.bbamcr.2015.04.005

Figure Lengend Snippet: Fig. 6. Ionomycin treatment in presence of EGTA does not show a SK1-induced caveolar microdomain. The cells were perfused with HBSS containing 150 μM EGTA followed by perfusion with EGTA and 5 μM ionomycin. Representative traces (A) and bar diagrams indicating the mean with S.E.M. (B) are shown, n = 3, ns = not significant.

Article Snippet: The lentiviral particles for SK1 shRNA (sc156038-V), as well as the control vectors (sc-108080), were acquired from Santa Cruz Biotechnology and the cells were transduced by incubating the cells for 12 h with polybrene (8 mg/ml) along with the lentiviral particles at multiplicity of infection 30.

Techniques:

Journal: Biochimica et biophysica acta

Article Title: A novel chimeric aequorin fused with caveolin-1 reveals a sphingosine kinase 1-regulated Ca²⁺ microdomain in the caveolar compartment.

doi: 10.1016/j.bbamcr.2015.04.005

Figure Lengend Snippet: Fig. 7. Store-operated Ca2+ entry (SOCE) at the caveolae and at the plasma membrane upon SK1 overexpression and downregulation, respectively. The cells were pre-treated with 1 μM thapsigargin (TG) in HBSS containing EGTA (150 μM) for 4 min. Then, the cells were perfused with 1 mM Ca2+ in the presence of 1 μM TG. SOCE was not affected by SK1 overexpression at the caveolae (A, B) nor at the PM (C, D), whereas knock-down of SK1 resulted in diminished SOCE specifically at the caveolae (E, F) but not at the overall PM (G, H). The bar diagrams indicate the means with S.E.M., n = 3–6, *P b 0.05.

Article Snippet: The lentiviral particles for SK1 shRNA (sc156038-V), as well as the control vectors (sc-108080), were acquired from Santa Cruz Biotechnology and the cells were transduced by incubating the cells for 12 h with polybrene (8 mg/ml) along with the lentiviral particles at multiplicity of infection 30.

Techniques: Clinical Proteomics, Membrane, Over Expression, Knockdown