Figure 5.

Anti-WNK1 antibody delays and inhibits plasma membrane PIP₂ re-synthesis following receptor-stimulated hydrolysis. HeLa cells transfected with M3R and GFP-tagged PLC-δ1-PH domain were microinjected with control IgG or anti-WNK1 antibody and stimulated by 1 µM CCh. (A) Changes in fluorescence intensity at the plasma membrane (decrease) and cytosol (increase) in response to carbachol were normalized by setting maximal change to 1.0. Data are means ± SEM of 18 and 20 cells (7 separate experiments) that were microinjected with control rabbit IgG or anti-WNK1 antibody, respectively. (B) Ratio of fluorescence intensity in the plasma membrane to that in cytosol from panel A. Lines indicate slopes of the initial recovery rates (0.032 ± 0.002 and 0.014 ± 0.002 for control IgG and anti-WNK1 antibody, respectively; p<0.01). (C) Representative confocal images (obtained across the middle of cells) from one of 7 experiments showing fluorescence at the plasma membrane and cytosol before addition of CCh and at 45 s (peak), 90 s and 180 s after addition of CCh. Signal intensity is shown by pseudo-color.