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. 2014 Jun 1;25(11):1769–1781. doi: 10.1091/mbc.E13-05-0292

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© 2014 Giurisato et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 3: — KSR2 depletion reduces SOCE. Immunoblot analysis showing the reduction of KSR2 (A) or KSR1 (B) in lysates from shRNA KSR2 HEK-293T, and shRNA control HEK-293T (Scr) cells. α-Tubulin (α-Tub) was used as loading control. (C) Spectrofluorimeter analysis of Ca²⁺ influx in indicated shRNA HEK-293T cells loaded with Fura-2. Ca²⁺ stores were depleted with 0.2 μM Tg (in Ca²⁺-free medium), and subsequently 1.2 mM Ca²⁺ was added to the medium to reveal Ca²⁺ influx. (D) Immunoblot analysis of KSR2 and STIM1 in lysates from shRNA KSR2 HeLa and shRNA control HeLa (Scr) cells. (E) Immunoblot analysis of KSR2 and STIM1in lysates from shRNA control HeLa (Scr) transfected with 0.2 μg of Flag or shRNA KSR2 HeLa transfected with 0.2 μg of KSR2-Flag. Lysates were blotted for KSR2 and STIM1. (F) Spectrofluorimeter analysis of Ca²⁺ influx in indicated shRNA HeLa cells loaded with Fura-2. Ca²⁺ influx was analyzed as in C. (G) Immunoblot analysis showing the presence of KSR2 in lysates from HEK-293T cells transfected as indicated. Lysates were blotted for KSR2. ERK1/2 was used as loading control. (H) Spectrofluorimeter analysis of 1 × 10⁶ HEK-293T cells transfected with expression vectors encoding KSR2-Flag at different doses (0.2, 0.4, 0.8 μg; top) or Flag alone as control (bottom). Elevation of [Ca²⁺]_i was measured with Fura-2 as described in C. Traces represent one of three independent experiments.