Figure 7: Cell-free kinase assay of PI3Kδ isoforms and small-molecule inhibition.

(a) Schematic representation of protein domains of PI3Kδ-L and -S isoforms. Adaptor (p85)-binding, RAS binding, C2, helix and catalytic domains are highlighted. Phosphorylation and ubiquitination sites (S, T, Y and K) and the region encoded by exon 20 (56 amino acids) residing in the catalytic domain are indicated. (b) Co-immunoprecipitation (Co-IP) of His-tagged PI3Kδ/p85α complex from transfected PC-3 cells followed by western blot, and (c) PI3K activity assays. S, supernatant; W, wash fraction; E, eluted fraction. Anti-His antibody was used in the Co-IP experiments, and anti-His, anti-p85α and anti-actin antibodies were used in the western blotting. *Significantly different kinase activities in total lysates of PI3Kδ-S versus PI3Kδ-L-expressing cells, or purified PI3Kδ-S/p85α versus PI3Kδ-L/p85α complexes. P<0.05 using two-sided Student’s t-test. Data presented as mean±s.e.m. of n=4 for each treatment group. (d) Purification of His-tagged PI3Kδ-L and -S isoforms. Western blot analysis of Ni-NTA resin-purified PI3Kδ isoforms from transfected PC-3 cells using His and PI3Kδ antibodies. FT, flow-through; W, wash fraction; E, eluted fraction. Closed arrowheads indicate PI3Kδ isoforms. (e) Cell-free kinase assay of L and S isoforms of PI3Kδ in the presence of vehicle (phosphate-buffered saline (PBS)), 100 nM wortmannin or 100 nM CAL-101. *Significantly different from vehicle control-treated PI3Kδ-L isoform. P<0.05 by ANOVA with Dunnett’s post hoc test. Data presented as mean±s.e.m. from at least four independent experiments for each treatment group. Blots in b,d are representative from at least three independent experiments with similar results. Variance was similar among groups being compared. Unprocessed western images are shown in Supplementary Fig. 13.