Panel A: Western blot analysis showing the interaction between EGFP-Endophilin-A1 and different regions of CAST. Three lanes display results for total lysate and precipitated proteins using anti-GFP and anti-RFP antibodies. Panel B: Western blot and Coomassie brilliant blue staining showing the direct binding of MBP-CAST-1-400 to various GST-tagged Endophilin-A proteins. Six lanes display input and pull-down results. Panel C: Western blot and Coomassie brilliant blue staining showing the effect of Myc-Dynamin-1-C on the binding of MBP-CAST-1-400 to GST-Endophilin-A1. Five lanes display input and pull-down results with varying amounts of Myc-Dynamin-1-C. Panel D: Fluorescence microscopy images showing the co-condensation of EGFP-Dynamin-1, ALFA-mCherry-CAST, and iRFP670-Endophilin-A2 in HEK293T cells. Images show different DNA transfection amounts and merged images with indicated arrows. Panel E: Fluorescence microscopy images showing the co-condensation of EGFP-Dynamin-1, ALFA-mCherry-CAST, and iRFP670-Endophilin-A2 BAR domain in HEK293T cells. Images show different DNA transfection amounts and merged images with indicated arrows.
Dynamin-mediated inhibition of CAST and endophilin-A binding. (A) N-terminal region of CAST binds to endophilin-A1. HEK293T cells expressing EGFP–endophilin-A1 were precipitated with ALFA-mCherry, ALFA-mCherry-CAST-1-400, or ALFA-mCherry-CAST-401-957 with recombinant ALFA nanobody-GST protein-binding GSH-Sepharose. Precipitated EGFP–endophilin-A1 was detected by western blot analysis. (B) Direct binding of CAST to endophilin-A family proteins. Recombinant protein of MBP-CAST-1-400 was precipitated with GST-endophilin-A1-full, GST-endophilin-A2-full, GST-endophilin-A2-BAR, or GST-endophilin-A3-BAR. MBP-CAST-1-400 was detected by western blot analysis and GST-endophilin-A proteins were detected by Coomassie brilliant blue (CBB) staining. (C) Dynamin-mediated inhibition of CAST and endophilin-A binding. MBP-CAST-1-400 and different amounts of Myc-dynamin-1-C (residues 657–867) were precipitated with GST-endophilin-A1-full. MBP-CAST-1-400 and Myc-dynamin-1-C were detected by western blot analysis and GST-endophilin-A1-full was detected by CBB staining. (D) HEK293T cells were transfected with EGFP-dynamin-1 (top panels; left panels; none of DNA; middle panels 0.01 μg of DNA; right panels 0.1 μg of DNA), ALFA-mCherry-CAST (second panels from the top), and iRFP670–endophilin-A2-expressing plasmid DNA (third panels from the top). Merged images show bottom panels, and white dotted arrow indicates co-condensation of ALFA-mCherry-CAST and iRFP670-endophilin-A2; green arrowhead indicates co-condensation of EGFP–dynamin-1 and iRFP670–endophilin-A2, and magenta arrow indicates condensation of ALFA-mCherry-CAST. Scale bar: 10 μm. (E) HEK293T cells were transfected with EGFP–dynamin-1 (top panels; left panels; none of DNA; middle panels 0.01 μg of DNA; right panels 0.1 μg of DNA), ALFA-mCherry-CAST (second panels from the top), and iRFP670–endophilin-A2 BAR domain–expressing plasmid DNA (third panels from the top). Merged images show bottom panels, and white dotted arrow indicates co-condensation of ALFA-mCherry-CAST, and iRFP670–endophilin-A2 BAR domain. Scale bar: 10 μm. Source data are available for this figure: SourceData F4.