![Figure 5. SGK1-dependent Orai1 membrane abundance and SOCE in megakaryocytic cell line MEG-01. (A) Confocal microscopy of Orai1 membrane abundance in nontransfected (control plasmid), S422DSGK1-transfected, and K127NSGK1-transfected MEG-01 cells (left). Red represents actin; green, Orai1; and blue, nuclei. Scale bar represents 10 μm. Statistical analysis of Orai1 immunofluorescence membrane abundance (right). *P < .05. **P < .01. n = 4. (B) Western blot analysis of Orai1 membrane abundance in nontransfected (control plasmid), S422DSGK1-transfected, and K127NSGK1-transfected MEG-01 cells. Arithmetic mean ± SEM (n = 7) of Orai1 protein abundance. *P < .05. **P < .01. (C) SOCE in SGK1-transfected MEG-01 cells. Fura-2-fluorescence reflecting cytosolic Ca2+ concentration [Ca2+]i of MEG-01 cells transfected with control-plasmid (gray), S422DSGK1 (black), or K127NSGK1 (red) after exposure to 5μM thapsigargin (Ca2+ store depletion) in the nominal absence of extracellular Ca2+ for 10 minutes and subsequent addition of 1mM extracellular Ca2+. Representative tracings (top) and arithmetic mean (bottom) of maximal Δ[Ca2+]i ± SEM (n = 15 per group) before and after addition of 1mM Ca2+. **P < .01. (D) Western blot analysis of Orai1 membrane abundance in nontransfected (control plasmid) MEG-01 cells and in S422DSGK1-transfected MEG-01 cells treated with the SGK1 inhibitor GSK650394 (1μM) or DMSO as solvent control. Arithmetic mean ± SEM (n = 4) of Orai1 protein abundance. **P < .01. (E) SOCE in MEG-01 cells treated with the specific SGK1 inhibitor GSK650394. Fura-2 fluorescence reflecting cytosolic Ca2+ concentration [Ca2+]i of MEG-01 cells transfected with control plasmid (gray) or S422DSGK1-transfected MEG-01 cells after treatment with GSK650394 (1μM, red) or DMSO (black) as solvent control. Representative tracings (top) and arithmetic mean (bottom) of maximal Δ[Ca2+]i ± SEM (n = 9 per group) after exposure to 5μM thapsigargin (Ca2+ store depletion) in the nominal absence of extracellular Ca2+ for 10 minutes and subsequent addition of 1mM extracellular Ca2+. **P < .01.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/119/1/10.1182_blood-2011-06-359976/4/zh89991183130005.jpeg?Expires=1724282151&Signature=rXmnJPktd7z06DSmo8LYbrI3-cn~VKeR-djaOrftXoq3cfZYNvv0ohgeoY~rzriTdeerOskLZoCRM-t-5tARRDtiDEXdE2RL6ygGlz9L5xqFJSvjDpaKK29PXxPKMTtYgG88g77J0cYcn-ZKeCgFgc~PI3iTYkmi5usiyyCLbEYXf~NqiUv3ARw~VdFAr~oBYOWOPuApJzih~ipXvwrCY1AX0h3wM8AA5Hssz408GogOuXeHTRDy6n529j9du5NyTbPjjz2tdFaW~fTzVkOkhTez4Ka2GkKGiKaoSmK~7D2PnB~P5v1InfrevejxEI7AQhmg7sjvRxOyXxGyTB042A__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
SGK1-dependent Orai1 membrane abundance and SOCE in megakaryocytic cell line MEG-01. (A) Confocal microscopy of Orai1 membrane abundance in nontransfected (control plasmid), S422DSGK1-transfected, and K127NSGK1-transfected MEG-01 cells (left). Red represents actin; green, Orai1; and blue, nuclei. Scale bar represents 10 μm. Statistical analysis of Orai1 immunofluorescence membrane abundance (right). *P < .05. **P < .01. n = 4. (B) Western blot analysis of Orai1 membrane abundance in nontransfected (control plasmid), S422DSGK1-transfected, and K127NSGK1-transfected MEG-01 cells. Arithmetic mean ± SEM (n = 7) of Orai1 protein abundance. *P < .05. **P < .01. (C) SOCE in SGK1-transfected MEG-01 cells. Fura-2-fluorescence reflecting cytosolic Ca2+ concentration [Ca2+]i of MEG-01 cells transfected with control-plasmid (gray), S422DSGK1 (black), or K127NSGK1 (red) after exposure to 5μM thapsigargin (Ca2+ store depletion) in the nominal absence of extracellular Ca2+ for 10 minutes and subsequent addition of 1mM extracellular Ca2+. Representative tracings (top) and arithmetic mean (bottom) of maximal Δ[Ca2+]i ± SEM (n = 15 per group) before and after addition of 1mM Ca2+. **P < .01. (D) Western blot analysis of Orai1 membrane abundance in nontransfected (control plasmid) MEG-01 cells and in S422DSGK1-transfected MEG-01 cells treated with the SGK1 inhibitor GSK650394 (1μM) or DMSO as solvent control. Arithmetic mean ± SEM (n = 4) of Orai1 protein abundance. **P < .01. (E) SOCE in MEG-01 cells treated with the specific SGK1 inhibitor GSK650394. Fura-2 fluorescence reflecting cytosolic Ca2+ concentration [Ca2+]i of MEG-01 cells transfected with control plasmid (gray) or S422DSGK1-transfected MEG-01 cells after treatment with GSK650394 (1μM, red) or DMSO (black) as solvent control. Representative tracings (top) and arithmetic mean (bottom) of maximal Δ[Ca2+]i ± SEM (n = 9 per group) after exposure to 5μM thapsigargin (Ca2+ store depletion) in the nominal absence of extracellular Ca2+ for 10 minutes and subsequent addition of 1mM extracellular Ca2+. **P < .01.
