Fig. 5.
Duox1-mediated regulation of intracellular Ca2+.(A) TCR-induced Ca2+ flux in Jurkat cell clones stably transfected with NT (dotted lines) or Duox1-specific (solid lines) shRNAs. Cells were loaded with Fluo-3 and Fura Red, stimulated with ionomycin or soluble IgM against TCR in the presence (+Ca2+) or absence (−Ca2+) of extracellular Ca2+, and immediately analyzed by flow cytometry. The data are presented as the ratio of Fluo-3 to Fura Red fluorescence over time and are representative of five separate experiments. (B) Association of IP3R1 with Duox1 in Jurkat cells. Duox1 was immuno-precipitated from lysates of Jurkat cells that were unstimulated or stimulated by TCR cross-linking for 5 min, and immunoprecipitates were analyzed by Western blotting for the presence of IP3R1 or Duox1. (C) IP3R1 coimmunoprecipitates with Duox1, but not Duox2, in stably transfected HEK 293 cells. Flp-In 293 clones stably expressing HA-tagged Duox1 or HA-tagged Duox2 were generated and incubated in the presence or absence of thapsigargin (TG). HA-tagged proteins were immunoprecipitated from the respective cell lysates and analyzed by Western blotting with antibodies against IP3R1 or the HA tag. (D) Whole-cell lysates (WCL) from the stable clones were analyzed by Western blotting for the presence of HA, Duox1, and β-tubulin as loading controls.