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Fig. 2. MTMR3 shows lipid binding associated with its PH-GRAM, but not its FYVE domain. (A) Lipid overlay assay. The isolated GST-MTMR3-FYVE domain does not present specific binding. As a control, equimolar GST-EEA1-FYVE domain demonstrates specific binding to Ptdins3P. (B) Binding of 3H-labelled liposomes to immobilised GST-FYVE domains. In contrast to GST-EEA1-FYVE, the GST-MTMR3-FYVE domain does not show PtdIns3P-dependent binding to liposomes. Black bars correspond to +1% PtdIns3P; white bars, no PtdIns3P. Results are the mean of duplicate points from a representative experiment. (C) The MTMR3 PH-G domain binds phosphoinositide lipids. Full length GST-MTMR3 (top panel), GST-MTMR3 ({Delta}PH-G) (centre) or GST-2 xPH-G (bottom) were tested for lipid binding in an overlay assay. (D) Liposome binding assay. Liposomes containing 0.5 or 0.25 mol% of the indicated phosphoinositides were tested for binding to GST-2 xPH-G. Blots were developed using anti-GST HRP-coupled antibody. MTMR3-PH-G domain showed increased selectivity for PtdIns5P phosphoinositide at reduced mole fractions (pellets, lower panels).