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. 2011 Jun;25(6):2012–2021. doi: 10.1096/fj.10-178210

Figure 3.

Figure 3.

Effect of ubiquitin ligase Nedd4-2 on Orai1 protein abundance and SOCE in HEK293 cells expressing Orai1 and STIM1. A) Confocal microscopy of Orai1 protein abundance in HEK293 cells stably expressing Orai1, transfected with control plasmid, Nedd4-2, or Nedd4-2/S422DSGK1. B) Arithmetic means of Orai1 protein abundance, determined by confocal microscopy in HEK293 cells stably expressing Orai1, transfected with control plasmid, Nedd4-2, or Nedd4-2/S422DSGK1 (n=4 independent experiments; 9 cells/experiment). C) Orai1 protein abundance of whole-lysate protein in HEK293 cells transfected with scrambled siRNA (control) or siRNA for Nedd4-2. Left panel: representative blot. Right panel: arithmetic means (n=4). D) Orai1 protein abundance of whole-cell lysate protein in HEK293 cells transfected with control plasmid, Nedd4-2, or Nedd4-2 and S422DSGK1. Left panel: representative blot. Right panel: arithmetic means (n=5). E) Arithmetic means (n=14–27) of peak and slope increase of Fura-2 fluorescence ratio in cells expressing Orai1 alone, Orai1/STIM1, Orai1/STIM1/S422DSGK1, Orai1/STIM1/Nedd4-2, or Orai1/STIM1/Nedd4-2/S422DSGK1. F) Geometric means (n=6) of change in the fluo3/fura red fluorescence ratio for HEK293 control cells (light shaded bar), STIM1-transfected cells (solid bar), and cells transfected with STIM1 and Nedd4-2 (dark shaded bar). Values are means ± se. *P < 0.05, **P < 0.01, ***P < 0.001; ANOVA.