Figure 2: IC87114-induced increase in PtdIns(4,5)P2 levels is specific to PI3Kδ and dependent on PTEN activity.
Mouse chromaffin cells were isolated from (a) wild-type (WT) littermate or (b) p110 δD910A/D910A mutant mice and treated with vehicle or IC87114 (1 μM) for 5 min before extracting phosphoinositides with acidified chloroform:methanol extraction and probing for PtdIns(4,5)P2 using the anti-PtdIns(4,5)P2 antibody, 2C11. Representative TLC blots from 4–6 independent experiments are shown. (c) Cultured bovine chromaffin cells were pre-incubated with or without VO-OHpic (100 nM) in buffer A for 20 min and subsequently treated for 5 min with vehicle alone or IC87114 (1 μM). Phosphoinositides were extracted as described above and PtdIns(4,5)P2 levels detected using the 2C11 antibody. A representative example of a TLC blot from 3 independent experiments is shown. Quantification of the change in PtdIns(4,5)P2 levels was achieved by densitometry analysis as described in the Methods section, and values are expressed as mean±s.e.m. (n=6 for WT, n=4 for p110 δD910A/D910A, n=3 for bovine chromaffin cells, Student's t-test, *P<0.05). (d,e) Chromaffin cells were transfected with PH-PLCδ1-EGFP, and plasma membrane patches were examined by TIRF microscopy at 1 frame per second. Images are displayed in pseudocolour to highlight the changes in fluorescence intensity in the indicated conditions. The images shown were taken from the beginning (left) and peak (right) of the IC87114-induced effect. The insets display the fluorescence intensity change from a selected region of interest on the plasma membrane patches taken at the indicated time points. (f) Time-course variation of PH-PLCδ1-EGFP fluorescence intensity on the (selected) plasma membrane patches in the indicated conditions. (g) Average peak fluorescence intensity change of PH-PLCδ1-EGFP in the respective conditions expressed as mean±s.e.m. (Student's t-test, **P<0.01). Scale bar, 10 μm.
