![Figure 6. SGK1-sensitive NF-κB–dependent transcription in MEG-01 cells und primary megakaryocytes. (A) Arithmetic mean ± SEM (n = 4) of mRNA encoding Orai1 in platelets (left) and megakaryocytes (right) from sgk1+/+ (black bar) and sgk1−/− mice (gray bar). **P < .01. (B) Arithmetic mean ± SEM (n = 6) of mRNA encoding Orai1 in nontransfected (control plasmid), S422DSGK1-transfected, and K127NSGK1-transfected MEG-01 cells of Orai1 mRNA abundance. **P < .01. (C) Western blot analysis of phospho-IKKα/β in nontransfected (control plasmid), S422DSGK1-transfected, and K127NSGK1-transfected MEG-01 cells. Arithmetic mean ± SEM (n = 5) of IKKα/β phosphorylation. **P < .01. (D) Western blot analysis of phospho-IκBα in nontransfected (control plasmid), S422DSGK1-transfected, and K127NSGK1-transfected MEG-01 cells. Arithmetic mean ± SEM (n = 5) of IκBα phosphorylation. **P < .01. (E) Arithmetic mean ± SEM (n = 6) of mRNA encoding Orai1 in nontransfected (control plasmid) and S422DSGK1-transfected MEG-01 cells incubated with SGK1 inhibitor GSK650394 (1μM), IKK inhibitor BMS-345541 (10μM), or DMSO as solvent control. **P < .01. (F) SOCE in MEG-01 cells treated with the highly specific IKK inhibitor BMS-345541. Fura-2-fluorescence reflecting cytosolic Ca2+ concentration [Ca2+]i of MEG-01 cells transfected with control-plasmid (gray), S422DSGK1-transfected MEG-01 cells treated with the IKK inhibitor BMS-345541 (10μM, red), or DMSO as solvent control (black) 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 (left) and arithmetic mean (right) of maximal Δ[Ca2+]i ± SEM (n = 8 per group) before and after addition of 1mM Ca2+. **P < .01. (G) Confocal microscopy of nuclear translocation of the NF-κB subunit p65 (RelA) in murine megakaryocytes cultivated from bone marrow of sgk1+/+ (top) and sgk1−/− (bottom) mice. Red represents GPIb; green, p65; and blue, nuclei. White arrows point to nuclear translocated p65. Bar represents 10 μm.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/119/1/10.1182_blood-2011-06-359976/4/zh89991183130006.jpeg?Expires=1724282472&Signature=iK4d3kkYsjQVHOR2QUVvTM-V-8CL-J6UFH8RCfGMo6KYBTeO2T9yekt1t542jEzZjE3pZaiL8wmsOmz0sJRlg1y8WuZOBiJX~E3gzwLvSz7-Ssni4qMJI7aKp6CJcm3aGb-XmccMMzJPlpObL4T9opK9QGS5-WQAMMagcUzt81bkVOzPYocK7yNWrUhTmdPxhei54Ay9MJdleyqwlnaTzp-UQHc95RBPjrMq65t25SRJm~W5Ju8b9BKECPpIUGcbzeML879WsywpMN6yPSs-OSGwcPkYbyyMLrij5M4rhrDqRxXfcJL5zI~ooRqi~72iPyZhXisjUqEL03tpspPa1g__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
SGK1-sensitive NF-κB–dependent transcription in MEG-01 cells und primary megakaryocytes. (A) Arithmetic mean ± SEM (n = 4) of mRNA encoding Orai1 in platelets (left) and megakaryocytes (right) from sgk1+/+ (black bar) and sgk1−/− mice (gray bar). **P < .01. (B) Arithmetic mean ± SEM (n = 6) of mRNA encoding Orai1 in nontransfected (control plasmid), S422DSGK1-transfected, and K127NSGK1-transfected MEG-01 cells of Orai1 mRNA abundance. **P < .01. (C) Western blot analysis of phospho-IKKα/β in nontransfected (control plasmid), S422DSGK1-transfected, and K127NSGK1-transfected MEG-01 cells. Arithmetic mean ± SEM (n = 5) of IKKα/β phosphorylation. **P < .01. (D) Western blot analysis of phospho-IκBα in nontransfected (control plasmid), S422DSGK1-transfected, and K127NSGK1-transfected MEG-01 cells. Arithmetic mean ± SEM (n = 5) of IκBα phosphorylation. **P < .01. (E) Arithmetic mean ± SEM (n = 6) of mRNA encoding Orai1 in nontransfected (control plasmid) and S422DSGK1-transfected MEG-01 cells incubated with SGK1 inhibitor GSK650394 (1μM), IKK inhibitor BMS-345541 (10μM), or DMSO as solvent control. **P < .01. (F) SOCE in MEG-01 cells treated with the highly specific IKK inhibitor BMS-345541. Fura-2-fluorescence reflecting cytosolic Ca2+ concentration [Ca2+]i of MEG-01 cells transfected with control-plasmid (gray), S422DSGK1-transfected MEG-01 cells treated with the IKK inhibitor BMS-345541 (10μM, red), or DMSO as solvent control (black) 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 (left) and arithmetic mean (right) of maximal Δ[Ca2+]i ± SEM (n = 8 per group) before and after addition of 1mM Ca2+. **P < .01. (G) Confocal microscopy of nuclear translocation of the NF-κB subunit p65 (RelA) in murine megakaryocytes cultivated from bone marrow of sgk1+/+ (top) and sgk1−/− (bottom) mice. Red represents GPIb; green, p65; and blue, nuclei. White arrows point to nuclear translocated p65. Bar represents 10 μm.
