Figure 1.
Leep2A and Leep2B localize to macropinocytic cups and nascent macropinosomes. (A) Mass spectrometry (MS) scores of Leep2A (DDB_G0284825) at the indicated time points following cAMP stimulation. Translocation score (the sum of protein scores at 10 and 20 s divided by the sum of all four time points) of Leep2A was calculated to be 0.61. (B) Quantification of GFP-Leep2A translocation in WT cells upon cAMP stimulation (1 μM cAMP was added at time 0; mean ± SD, n represents the number of cells analyzed). (C) Time-lapse imaging of GFP-Leep2A translocation in response to cAMP stimulation in WT cells (1 μM cAMP was added at time 0). (D) Localization of GFP-Leep2A in WT cells during macropinocytosis. Arrowhead indicates an emerging macropinosome. (E) Localization of GFP-Leep2A in WT cells during random migration. Arrowhead indicates the migrating front. (F) Proteomic identification of Leep2B (DDB_G0281809) as a binding partner of Leep2A. GFP-Leep2A was immunoprecipitated from cell lysates by GFP-trap and the bound proteins analyzed by mass spectrometry. PSMs, peptide spectrum matches. (G) Co-IP of RFP-Leep2B or PHcrac-RFP with GFP or GFP-Leep2A. Fluorescent fusion proteins were expressed in WT cells. IP was performed with GFP-trap and samples were probed with GFP or RFP antibody. (H) Localization of GFP-Leep2B in WT cells during macropinocytosis. Arrowhead indicates an emerging macropinosome. (I) Colocalization of GFP-Leep2A and RFP-Leep2B in WT cells. Arrowheads indicate an emerging macropinosome. (J) Localization of GFP-Leep2A and RFP-Leep2B in WT cells migrating under agarose along a folic acid (FA) gradient. The white triangle indicates the gradient direction. Scale bars, 5 μm. Source data are available for this figure: SourceData F1.

Leep2A and Leep2B localize to macropinocytic cups and nascent macropinosomes. (A) Mass spectrometry (MS) scores of Leep2A (DDB_G0284825) at the indicated time points following cAMP stimulation. Translocation score (the sum of protein scores at 10 and 20 s divided by the sum of all four time points) of Leep2A was calculated to be 0.61. (B) Quantification of GFP-Leep2A translocation in WT cells upon cAMP stimulation (1 μM cAMP was added at time 0; mean ± SD, n represents the number of cells analyzed). (C) Time-lapse imaging of GFP-Leep2A translocation in response to cAMP stimulation in WT cells (1 μM cAMP was added at time 0). (D) Localization of GFP-Leep2A in WT cells during macropinocytosis. Arrowhead indicates an emerging macropinosome. (E) Localization of GFP-Leep2A in WT cells during random migration. Arrowhead indicates the migrating front. (F) Proteomic identification of Leep2B (DDB_G0281809) as a binding partner of Leep2A. GFP-Leep2A was immunoprecipitated from cell lysates by GFP-trap and the bound proteins analyzed by mass spectrometry. PSMs, peptide spectrum matches. (G) Co-IP of RFP-Leep2B or PHcrac-RFP with GFP or GFP-Leep2A. Fluorescent fusion proteins were expressed in WT cells. IP was performed with GFP-trap and samples were probed with GFP or RFP antibody. (H) Localization of GFP-Leep2B in WT cells during macropinocytosis. Arrowhead indicates an emerging macropinosome. (I) Colocalization of GFP-Leep2A and RFP-Leep2B in WT cells. Arrowheads indicate an emerging macropinosome. (J) Localization of GFP-Leep2A and RFP-Leep2B in WT cells migrating under agarose along a folic acid (FA) gradient. The white triangle indicates the gradient direction. Scale bars, 5 μm. Source data are available for this figure: SourceData F1.

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