Fig. 1.
Generation and characterisation of C2αD1268A/WT KI mice. (a) Gene targeting strategy to introduce the D1268A mutation in the DFG motif in exon 24 of the Pik3c2a gene. The FRT-flanked cassette encoding the Pgk Neo selection marker was removed in vivo by breeding onto ACTB-Flp mice. (b) PI3K-C2α protein expression. Tissue homogenates were analysed by SDS–PAGE and immunoblotting using anti-PI3K-C2α antibody. (c) PI3K isoform expression in WT and C2αD1268A/WT cells and tissue. Each lane on the SDS–PAGE gel represents an independent mouse. Homogenates of MEFs or epididymal WAT from male mice were analysed by SDS–PAGE and immunoblotting using the indicated antibodies. (d) Lipid kinase activity associated with PI3K-C2α in WT and C2αD1268A/WT mice. Homogenates of MEFs or epididymal WAT from male mice were immunoprecipitated using PI3K-C2α antibody, and subjected to an in vitro PI3K activity assay. Results shown are pooled data from three independent experiments (each with 2–4 experimental replicates). *p < 0.05; ***p < 0.001