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Fig. 3. AMER1 localizes to the membrane via binding to PtdIns(4,5)P₂. (A) Double staining of EGFP, EGFP-AMER1, EGFP-AMER1 deletion constructs as in Fig. 2E or EGFP-AMER2 (upper panels, GFP fluorescence), and APC (lower panels, antibody Ali immunofluorescence) in MCF-7 cells transiently transfected as indicated above the panels. Arrowheads denote the membrane, and arrows the filamentous localizations. (B) Membrane lipid-binding assays of AMER1 deletion mutants. Membrane lipid strips were incubated with the indicated recombinant GST-AMER1 fusion proteins, revealing two phosphoinositide binding sites. DAG, diacylglycerol; PA, phosphatidic acid; PS/PE/PC/PG, phosphatidyl-serine/-ethanolamine/-choline/-glycerol. (C) Localization of AMER1 (a-e) and APC (a'-c') or AMER1 deletion mutants (f-k) in transiently transfected MCF7 cells. Double staining of EGFP-AMER1 (a-c, GFP fluorescence) and APC (a'-c', anti-M-APC immunofluorescence) with (b,b') and without (a,a') prior ionomycin treatment (Iono), or with ionomycin treatment followed by EGTA treatment (c,c'). Staining of EGFP-AMER1 in cells treated with wortmannin (Wm) prior to ionomycin/EGTA treatment (d) or with neomycin (Neo) prior to ionomycin (e). Staining of EGFP-AMER1(2-142) (f-h) or EGFP-AMER1(143-209) (i-k) in cells treated with ionomycin/EGTA as indicated.

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