Figure 6
From: Dynamic interaction of SARAF with STIM1 and Orai1 to modulate store-operated calcium entry
Analysis of the Orai1 site that interacts with SARAF.
NG115-401L cells were transfected with SARAF alone or in combination with either pEYFP-full-length Orai1 (FL-Orai1), the N-terminal deletion mutants (pEYFP-Orai1 ΔN1–38 (Orai1 ΔN1–38), ΔN1–72 (Orai1 ΔN1–72) and ΔN1–89 (Orai1 ΔN1–89) (a), pEYFP-Orai1 C-terminal deletion mutant (Orai1-ΔCtermin, amino acids 1–260 (b,c) or empty vector (Mock), as indicated. After 48 h cells were lysed and the whole cell lysates were immunoprecipitated (IP) with anti-SARAF antibody (a,b) or anti-YFP antibody (c). Immunoprecipitates were subjected to 10% SDS-PAGE and subsequent Western blotting with a specific anti-Orai1 (amino acids 288–301, Sigma) antibody (a), anti-Orai1 (ab177021, Abcam) antibody (b) or anti-SARAF antibody (c). Membranes were reprobed with the antibody used for immunoprecipitation for protein loading control (middle panels). (a,b) Alternatively, the cell lysates were subjected to 10% SDS-PAGE and subsequent Western blotting with anti-Orai1 antibody (specific for amino acids 288–301; panel a, bottom) or with anti-Orai1 (ab177021, Abcam; panel b, bottom). HC, heavy chain of the antibody used for immunoprecipitation. The panels show results from one experiment representative of 3 others. Molecular masses indicated on the right were determined using molecular-mass markers run in the same gel.
