Journal: The EMBO Journal
Article Title: CHC22 clathrin recruitment to the early secretory pathway requires two-site interaction with SNX5 and p115
doi: 10.1038/s44318-024-00198-y
Figure Lengend Snippet: ( A , B ) Representative immunoblot ( n = 4–6) of immunoprecipitates (IP) of CHC22, from control HeLa cells ( A ) and differentiated human skeletal muscle cell line AB1190 (Myotubes) ( B ). Samples were immunoblotted for CHC22, SNX5, SNX6, and tubulin. Lanes show Input (5%), bead-only (no antibody) control (Bead) and CHC22 immunoprecipitate (22 IP). The migration positions of MW markers are indicated at the left in kilodaltons (kD). ( C ) The structure of a single clathrin triskelion formed from three clathrin heavy chains with the Hub region indicated by red dotted triangle and the trimerization domain (TxD), magenta), proximal leg region (dark blue), knee and distal leg regions (grey) and terminal domain (light blue), based on PDB 31YV (Fotin et al, ). ( D ) AlphaFold-generated model of the interaction between CHC22 Hub (blue proximal leg residues 1278–1519 and magenta TxD residues 1520–1640) and the BAR domain of SNX5 (residues 202–404, orange). Two angles of view are displayed. ( E ) Representative immunoblot ( n = 3–5) of in vitro binding of full-length GST-SNX5 (right lanes) or GST alone (left lanes) to CHC22 Hub (22 Hub) or CHC17 Hub (17 Hub) immobilized on Ni-NTA beads. Purified protein input (2%) and bead-only (no Hub immobilized) control (Bead) are shown for each prey. Samples were immunoblotted for SNX5, His-tag or GST with detected proteins indicated at the right. The position of MW markers is indicated at the left in kD. (F) Representative immunoblot of in vitro binding of full-length GST-SNX5 (right lanes) or GST alone (left lanes) to CHC22 Hub or CHC22 TxD immobilized on Ni-NTA beads ( n = 3). Purified protein input (2%) and beads without CHC22 fragment added (Bead) are shown for each prey. Samples were immunoblotted for SNX5, His-tag or GST with detected proteins indicated at the right. The position of MW markers is indicated at the left in kD. .
Article Snippet: The human myoblast cell line AB1190 was grown in complete Skeletal Muscle Cell Growth Medium (Promocell) with provided supplemental cocktail and 10% FBS (Gibco) to reach a 15% final supplement concentration (volume/volume) (Camus et al, ).
Techniques: Western Blot, Control, Migration, Generated, In Vitro, Binding Assay, Purification
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