Coimmunoprecipitation of SARAF with SOAR and STIM1 mutants. The STIM1 proteins are tagged with YFP and SARAF with Myc. Anti-YFP and anti-Myc were used for detection of protein expression and coimmunoprecipitation. All proteins were expressed in HEK cells. (a) SARAF interacts with SOAR but not with disrupted SOAR(4K/4A). (b) STIM1 CC1 (233–342) and STIM1(445–550) that includes CTID do not interact with SARAF, whereas SOAR (STIM1(344–442)), STIM1(233–460), and STIM1(233–535) that include SOAR interact with SARAF. Controls were performed with each construct in the absence of precipitating antibodies. (c) The STIM1Δ447–460 and STIM1Δ475–490 mutations increased interaction whereas the STIM1Δ448–521 and STIM1Δ448–530 mutations markedly reduced binding of STIM1 with SARAF. (d) Coimmunoprecipitation of STIM1, Δ447–460, Δ475–490, and their 4K/4A mutants in SOAR, SOAR, and STIM1(344–485) with SARAF. The 4K/4A mutation markedly reduced the interaction with SARAF. One lane, which tested the effect of the 4E mutation on Δ475–490, marked by the gap was deleted for presentation purposes. The effect of this mutation is shown and is discussed in relation to Fig. 6 c. (e) Summary of the coimmunoprecipitation of the indicated deletions and number of experiments. The binding was normalized to input and used to calculate fold change relative to STIM1. Asterisk denotes P < 0.01 or better relative to STIM1.