Journal: Frontiers in Immunology
Article Title: Inhibition of Orai Channel Function Regulates Mas-Related G Protein-Coupled Receptor-Mediated Responses in Mast Cells
Figure Lengend Snippet: Lentiviral shRNA-Mediated Knockdown of Orai1, Orai2, and Orai3 Reduces SP-Induced Responses in LAD2 Cells. (A) Knockdown efficiency of each Orai channel was determined by western blotting in cells transduced with non-targeted (control) or Orai-specific shRNA and a representative blot is shown. (B) Quantification of Orai knockdown is represented as bar graph of relative intensities of Orai protein bands normalized to respective β-Actin. (C, E, G) Cells were loaded with Fura-2AM, resuspended in Ca 2+ -free buffer (1.5 ml) and measurement of intracellular Ca 2+ mobilization was initiated via the addition of SP (0.3 µM, at 100s). At 400 s, Ca 2+ concentration of the buffer was increased to 1 mM via the addition of 1.5 µl of 1 M calcium and influx was measured for additional 18 min. (D, F, H) Effects of Orai knockdown on SP-induced degranulation, as measured by β-hexosaminidase release. Data presented are mean ± SEM of N = 4 experiments. Statistical significance was determined by two-way ANOVA with Tukey’s multiple comparisons at a value ***p
Article Snippet: Rabbit anti-Orai1, Orai2 and Orai3 antibodies from Alomone lab (Rockville, MD, USA), anti-ERK1/2, anti-phospho-ERK1/2 (Thr-202/Tyr-204), anti-phospho-Akt (Ser-473), anti-Akt, β-Actin and goat anti-rabbit IgG-HRP were obtained from Cell Signaling Technology (Danvers, MA, USA).
Techniques: shRNA, Western Blot, Transduction, Concentration Assay