Journal: eLife

Article Title: Clathrin-independent endocytic retrieval of SV proteins mediated by the clathrin adaptor AP-2 at mammalian central synapses

doi: 10.7554/eLife.71198

Figure Lengend Snippet:

Article Snippet: Antibody , AP-2 α-adaptin(mouse monoclonal) , Santa Cruz , Cat# sc-55497RRID: AB_2056344 , WB: 1:100.

Techniques: Recombinant, shRNA, Transfection, Imaging, Software

Journal: The EMBO Journal

Article Title: ArfGAP1 inhibits mTORC1 lysosomal localization and activation

doi: 10.15252/embj.2020106412

Figure Lengend Snippet: RagA/B knockout (KO) human embryonic kidney 293A (HEK293A) cells were transfected with siRNAs against clathrin (CHC) or negative control siRNA (siCtrl) for 48 h and then co‐transfected with siRNAs and HA‐tagged ArfGAP1 or HA‐tagged RFP1 (control) with Myc‐tagged S6K1 for 48 h. Cells were starved of amino acids (−AA) for 2 h, and then, 4 mM of glutamine (+Gln) or asparagine (+Asn) was added for 2 h. Cell lysates were probed for HA, Myc, Actin, CHC, and mTORC1 activity assessed by the phosphorylation of S6K1 at threonine 389 (pS6K1). RagA/B KO HEK293A cells were transfected with siRNAs against clathrin (CHC) or negative control siRNA (siCtrl) for 72 h. Cells were starved of amino acids (−AA), and then, 4 mM of glutamine (+Gln) or asparagine (+Asn) was added for 2 h. mTORC1 activity was analyzed by phosphorylation of S6K1 at threonine 389 (pS6K1). Actin was probed for as a loading control. Protein levels of clathrin (CHC) were also confirmed by immunoblotting. Similar experiments were performed as in (A) except that siRNA against the µ subunit of AP2 (AP2M1) was used. Lysates were analyzed similarly to (A) except that protein levels of AP2 (α subunit) were confirmed by immunoblotting. RagA/B KO HEK293A cells were transfected with siRNAs against AP2 complex (AP2M1 or AP2A1 for the µ or α subunit, respectively) or negative control siRNA (siCtrl) for 72 h. Cells were treated and lysates analyzed similar to (C). Protein levels of AP2 (α subunit) were confirmed by immunoblotting. NC denotes normal condition. RagA/B KO HEK293A cells were transfected with HA‐tagged ArfGAP1, HA‐tagged ArfGAP2, or RFP1 (control) for 24 h. Cell lysates were immunoprecipitated with anti‐HA beads. Immunoprecipitates (IP) or whole cell lysate (WCL) samples were probed for HA and components of AP 1–5 complexes, and clathrin (CHC).

Article Snippet: AP‐1 (#sc‐398867, 1:1,000), AP‐2 Adaptin 1/2 (#sc‐17771, 1:2,000), AP‐3 (#sc‐136277, 1:1,000), AP‐4 (#sc‐135835, 1:1,000), CHC (#sc‐12734, 1:1,000), HA (#sc‐7392, 1:500) and Myc (#sc‐40, 1: 1,000) were obtained from Santa Cruz.

Techniques: Knock-Out, Transfection, Negative Control, Control, Activity Assay, Phospho-proteomics, Western Blot, Immunoprecipitation

Journal: eLife

Article Title: A nanobody-based molecular toolkit provides new mechanistic insight into clathrin-coat initiation

doi: 10.7554/eLife.41768

Figure Lengend Snippet:

Article Snippet: Antibody , anti-AP-2 α subunit mouse mAb C-8 , Santa Cruz Biotechnology , sc-17771 , 1:500 for immunoblotting.

Techniques: Western Blot, Immunofluorescence, Affinity Purification, Transduction, Sequencing, Plasmid Preparation, Control, Recombinant, Software