Figure 4

Time-course of TG-induced SARAF-STIM1 and SARAF-Orai1 co-immunoprecipitation.

MEG-01 cells (a) and NG115-401L cells (b) were stimulated with TG (1 μM) in the presence of 300 μM extracellular Ca²⁺. Samples were removed 30s before the addition of TG and 30, 60 and 120 s after the treatment with TG. Whole cell lysates were immunoprecipitated (IP) with the indicated antibody and immunoprecipitates were subjected to 10% SDS-PAGE and subsequent Western blotting with specific anti-Orai1 (aa 288–301 (Sigma)), anti-SARAF and anti-STIM1 antibodies. Membranes were reprobed with the antibody used for immunoprecipitation for protein loading control. The panels show results from one experiment representative of 5 others. Molecular masses indicated on the right were determined using molecular-mass markers run in the same gel. HC, heavy chain of the antibody used for immunoprecipitation. Data represent the quantification of SARAF-Orai1 and SARAF-STIM1 association in resting and TG-treated cells. Results are recorded as arbitrary optical density units, expressed as mean ± S.E.M. and presented as percentage of control (resting cells). * and *** represent p < 0.05 and p < 0.001, as compared to controls.