FIGURE 2:

KSR2 is required for SOCE. T- and B-lymphocytes (3 × 10⁶ cells) purified from wt (black line) or ksr2^−/− (red line) mice were loaded with Fura-2. The experiments were done in a spectrofluorimeter cuvette in a nominally Ca²⁺-free medium containing 0.2 mM EGTA. (A) Intracellular Ca²⁺ stores were depleted with 0.2 μM thapsigargin (Tg); subsequently 1.2 mM Ca²⁺ was added to the medium to reveal Ca²⁺ influx. (B) Elevation of [Ca²⁺]_i was induced by anti-CD3 (2C11) followed by anti-IgG antibody (anti-hamster) or anti-IgM stimulation for T- and B-lymphocytes, respectively. (C) Store-dependent Ca²⁺ influx was also evaluated from the rate of Fura-2 fluorescence quenching by Mn²⁺ (see Materials and Methods). Fura-2–loaded cells were resuspended in cuvette containing Ca²⁺-free medium and treated or not with agonists (2C11 + anti-hamster or anti-IgM for T- and B-cells, respectively). After 4 min, 0.1 mM MnCl₂ was added. Data (shown after MnCl₂ addition) were normalized as percentage of fluorescence values obtained immediately before addition of MnCl₂. Traces represent one of three independent experiments.