Abnormal STIM1 mRNA splicing and lack of protein expression in the patient’s EBV-B cells. (A) Levels of STIM1 mRNA were assessed by quantitative RT-PCR in the EBV-B cells from five healthy controls (C) and the patient (P). Threshold cycles (CT) for STIM1, normalized to those of GUS (ΔCT), are plotted as 2−(ΔCT). Each dot or square represents a mean of three independent experiments for each individual. The horizontal bar indicates the mean of five healthy controls. (B) STIM1 exons 7–9 were amplified from cDNA from EBV-B cells of the control (C) or the patient (P) and ligated to pCR2.1 vector. Clones containing STIM1 transcripts were sequenced, and the frequency of each splice variant was calculated by dividing the number of clones containing the particular transcript by the total number of sequenced clones (n = 12 for C, n = 120 for P). 16/120 of P clones corresponded to other minor abnormal transcript forms (not depicted). Number of base pairs indicated between the exons in abnormally spliced variants of the patient represents the size of gain or loss, compared with the wild-type transcript. (C) Levels of STIM1 protein were assessed by immunoblotting with an antibody against the C terminus (left) or N terminus (right) of STIM1. Two fibroblast cell lines, derived from either a healthy individual (C+) or a previously reported STIM1-deficient patient (C−; Picard et al., 2009), were used as controls. C1, C2, and C3 show EBV-B cells from healthy controls and P shows EBV-B cells from the patients. GAPDH blots show comparable protein loading for each sample. Asterisks indicate nonspecific protein bands. Representative blots of three independent experiments are shown.
