FIG. 4.
Membrane turnover of γc is independent of Jak3. EBV-transformed human B-cell lines from normal individuals (control) and Jak3-SCID patients were treated with either CHX (20 μg/ml) (A) or BFA (2 μg/ml) (B and C) for the time indicated, fixed, and stained with anti-human CD132-PE antibody to determine γc surface levels by flow cytometry. The experiment was done in triplicate for each time point. The MFI indicates the channel number in a linear scale, which corresponds to the MFIs obtained for a particular antibody. The ΔMFI was calculated for each sample by subtracting the geometric mean fluorescence of the corresponding isotype control from those relative to the CD132-specific antibody. Cells from patient 1 (in panels A and B) are homozygous for a missense mutation Y100→C (18); this mutant version of Jak3 is expressed but does not bind to γc (1, 36). Cells from patient 2 (C) are homozygous for a 151-bp deletion in the pseudokinase domain, leading to a frameshift and premature termination (18); this Jak3 variant is not expressed.