Activation of phosphatidylinositol 3-kinase (PI3K) is critical for interleukin 3 (IL-3)-mediated immunoglobulin A (IgA) binding.
IgA-binding studies were performed with Ba/F3_FcαR cells, co-expressing p21ras mutants (RasV12, V12S35, V12C40, or N17) (A), PI3K mutants (p110K227E or ▵p85) or active PTEN (B). Cells were cytokine-starved for 4 hours and treated with buffer (white bars) or IL-3 (1:1000; gray bars) for 15 minutes. Binding of IgA beads to the cells was measured, and results are expressed as rosette index (number of beads/100 cells) and as means ± SE (n = 4). (C) Cytokine-starved Ba/F3 stable cell lines were stimulated with or without IL-3 for 15 minutes. After stimulation, cells (0.2 × 106 per sample) were washed with ice-cold phosphate-buffered saline, lyzed in lysis buffer, and heated for 5 minutes after addition of 5x sample buffer. Phosphorylation STAT5 was detected, using polyclonal anti-phospho-STAT5 (Tyr694) antiserum for Western blotting.
