Fig. 3

DLC1-START binds Phosphatidylserine and this binding improves its binding to Caveolin-1 or PLCD1. A Pull-down (PD) using control (ctrl) or PS-coated beads in H358 (top) or 293 T (bottom) cells overexpressing the indicated GST or GFP-fusion plasmids, followed by GST or GFP Western blot (WB). The correct expression of all plasmids was verified in total lysates by WB. B Immunoprecipitation (IP) in 293 T overexpressing GST Caveolin-1 (GST CAV) (top) or GFP DLC1 (bottom) using the indicated antibodies, in the presence or absence of 100 μM PS added to the binding reaction. Immunocomplexes were analyzed by Western blot (WB) using the indicated antibodies. C Immunoprecipitation (IP) in 293 T overexpressing GST PLCD1 (GST PLCD1) (top) or GFP DLC1 (bottom) using the indicated antibodies, in the presence or absence of 100 μM PS added to the binding reaction. Immunocomplexes were analyzed by Western blot (WB) using the indicated antibodies. D Pull-down (PD) using MBP control or MBP DLC1 START 848–1091 beads in 293 T overexpressing the indicated GST-fusion plasmids, in the presence or absence of 100 μM PS added to the binding reaction, followed by GST Western blot. The amount of MBP-purified proteins is shown as stained with Ponceau S (bottom panel). E Pull-down (PD) using control (ctrl) or PS-coated beads and MBP control or MBP DLC1 START 848–1091 purified proteins (eluted from amylose resin in the presence of maltose), followed by MBP Western blot (WB). The purity and amount of MBP-purified proteins is shown in the western blot from the inputs, which represents a fraction of the total purified protein used in the pull-down