pdown gateway entry vectors  (VectorBuilder GmbH)

Journal: bioRxiv

Article Title: The TRIM9/TRIM67 neuronal interactome reveals novel activators of morphogenesis

doi: 10.1101/2020.10.02.323980

Figure Lengend Snippet: (A) Graphical representation of the BioID approach. E15.5 cortical neurons were transduced with HSV carrying Myc-BirA*, Myc-BirA*TRIM9ΔRING, or Myc-BirA*TRIM67ΔRING, media was supplemented with 50 μM biotin. Following cell lysis, biotinylated proteins were affinity purified, enriched proteins were subjected to on-bead trypsinization. Candidate peptides were identified by mass spectrometry. (B) Images of axonal growth cones from control Myc-BirA*, Myc-BirA*-TRIM9ΔRING, Myc-BirA*-TRIM67ΔRING expressing neurons at 0 and 24 hrs post-Biotin addition, stained with fluorescent streptavidin and anti-Myc antibody. Merged images show colocalized streptavidin and Myc signals. (C) Western blots of streptavidin affinity purification of biotinylated proteins from cortical neurons expressing Myc-BirA* or Myc-BirA*-TRIM9ΔRING. Samples include inputs and affinity purified samples from controls that do not express BirA* (6.5, 16, 24 hrs), input from neurons expressing Myc-BirA* but not supplemented with biotin, inputs (16, 24 hrs) and affinity purified samples (6.5, 16, 24 hrs) from neurons expressing Myc-BirA* and supplemented with biotin, affinity purified samples from neurons expressing Myc-BirA*-TRIM9ΔRING (6.5, 16, 24 hrs). Blots were probed for streptavidin, DCC, VASP.

Article Snippet: The pDOWN Gateway entry vectors for expressing Myc-BirA*, Myc-BirA*TRIM9ΔRING or Myc-BirA*TRIM67ΔRING were constructed by VectorBuilder.

Techniques: Transduction, Lysis, Affinity Purification, Mass Spectrometry, Expressing, Staining, Western Blot

Journal: bioRxiv

Article Title: The TRIM9/TRIM67 neuronal interactome reveals novel activators of morphogenesis

doi: 10.1101/2020.10.02.323980

Figure Lengend Snippet: (A-G) Immunoblots showing co-immunoprecipitation (Co-IP) of GFP- or HA-tagged proteins when immunoprecipitated using an anti-Myc antibody to enrich for Myc-TRIM9ΔRING or Myc-TRIM67ΔRING. (A: Coro1A, B: Sipa1l1, C: Myo16, D: Grip1a, E: Kif1a, F: ExoC1, G: VCP). A-G show blots for inputs for the co-immunoprecipitated proteins, the immunoprecipitated Myc-TRIM9ΔRING or Myc-TRIM67ΔRING and co-immunoprecipitated proteins. (H) Immunoblot demonstrating co-IP of Myc-TRIM67ΔRING, but not Myc-TRIM9ΔRING with GFP-MAP1B. Two representative blots are included, one with GFP-control co-expressed with Myc-TRIM9ΔRING and one with GFP-control co-expressed with Myc-TRIM67ΔRING. (I) Immunoblot showing Myc-TRIM9ΔRING and Myc-TRIM67ΔRING co-immunoprecipitating with GFP-LPPR4 (PRG1). The black arrow in the blot probed for GFP shows a faint GFP-LPPR4 band. Note that GFP-LPPR4 was not detected in inputs. (J(i), (ii)) Immunoblots demonstrating HA-Ywhae does not co-IP with Myc-TRIMΔRING or full-length constructs of TRIM9 and TRIM67. The white asterisks in J(i) denote a non-specific band observed in all anti-Myc IP lanes.

Article Snippet: The pDOWN Gateway entry vectors for expressing Myc-BirA*, Myc-BirA*TRIM9ΔRING or Myc-BirA*TRIM67ΔRING were constructed by VectorBuilder.

Techniques: Western Blot, Immunoprecipitation, Co-Immunoprecipitation Assay, Construct

Journal: bioRxiv

Article Title: The TRIM9/TRIM67 neuronal interactome reveals novel activators of morphogenesis

doi: 10.1101/2020.10.02.323980

Figure Lengend Snippet: (A) Representative images to demonstrate colocalization of GFP-Myo16 with tagRFP-TRIM67ΔRING. Zoomed inset and the blue arrowheads show colocalization along the axon and in filopodia along the axon. (B) Representative images to demonstrate colocalization of GFP-LPPR4 with tagRFP-TRIM67. Zoomed inset and the blue arrowheads show colocalization along the axon (yellow box) and at the axonal growth cone (red box).

Article Snippet: The pDOWN Gateway entry vectors for expressing Myc-BirA*, Myc-BirA*TRIM9ΔRING or Myc-BirA*TRIM67ΔRING were constructed by VectorBuilder.

Techniques:

Journal: bioRxiv

Article Title: The TRIM9/TRIM67 neuronal interactome reveals novel activators of morphogenesis

doi: 10.1101/2020.10.02.323980

Figure Lengend Snippet: (A) Immunoblot demonstrating GFP-Myo16 co-IP specifically with Class I TRIM proteins, TRIM9 and TRIM67, but not TRIM18 (TRIM proteins used in this assay are full-length and Myc-tagged). (B) Representative immunoblot of TRIM67 domain deletion constructs (TRIM67ΔRING, TRIM67ΔCC, TRIM67ΔFN3, TRIM67ΔSPRY) IP demonstrating that the FN3 domain of TRIM67 is necessary for GFP-Myo16 and Myc-TRIM67 interaction. (C) Representative images demonstrating endogenous localization of Myo16 in embryonic cortical neurons co-stained with phalloidin for filamentous actin and βIII tubulin to detect MTs. Myo16 localizes to the tips of filopodia and along the axon. (D) Immunoblot showing endogenous Myo16 protein levels were not significantly different in cultured embryonic cortical neurons from wildtype ( Trim9 +/+ : Trim67 +/+ ), Trim9 -/- : Trim67 +/+ , Trim9 +/+ : Trim67 -/- at 2 DIV. Quantification from 3 biological replicates. (E) Representative immunoblot demonstrating GFP (control) and GFP-Myo16 ubiquitination levels in TRIM67 -/- HEK cells expressing either Myc or Myc-TRIM67. The blot was probed with anti-GFP and anti-HA antibodies to visualize Myo16 and ubiquitin respectively. Myo16 ubiquitination status is not altered. (F) Representative immunoblot demonstrating GFP-Myo16 ubiquitination levels in wildtype, TRIM9 -/- ; TRIM67 +/+ , TRIM9 +/+ ; TRIM67 -/- HEK cells. The blot was probed with anti-GFP and anti-HA antibodies to visualize Myo16 and ubiquitin respectively. Myo16 ubiquitination status is not altered.

Article Snippet: The pDOWN Gateway entry vectors for expressing Myc-BirA*, Myc-BirA*TRIM9ΔRING or Myc-BirA*TRIM67ΔRING were constructed by VectorBuilder.

Techniques: Western Blot, Co-Immunoprecipitation Assay, Construct, Staining, Cell Culture, Expressing