Journal: bioRxiv

Article Title: N-Cadherin Provides a Cis and Trans Ligand for Astrotactin that Functions in Glial-Guided Neuronal Migration

doi: 10.1101/357541

Figure Lengend Snippet: Flow cytometry of live HEK 293T cells labeled with GFP and Alexa Fluor 647 antibodies. Control, untransfected cells (A) or cells transfected with Venus (B) , Astnl-FL-Venus (C) , or Astnl-ΔCTD-Venus , lacking the MACPF, FNIII and ANX-like domains in the C-terminus (D) . The x-axis shows total GFP fluorescence and the y-axis shows surface labeling (Alexa Fluor 647). Thus, cells expressing cytosolic Venus-tagged proteins are indicated in the lower right quadrant (Q3), while double positive cells (GFP+/Alexa Fluor 647+) in the upper right quadrant (Q2) express Venus-tagged proteins exposed on the cell surface. The percentage of cells expressing ASTN1 on the cell surface is depicted. Both ASTN1-FL and ASTNi-ΔCTD were significantly expressed on the cell surface (58 % and 49 % of live transfected cells, respectively).

Article Snippet: After removing the beads, the lysates were incubated with 3 μg of a rabbit GFP antibody (Invitrogen), rabbit Astn1 antibody, or normal rabbit IgG (Santa Cruz Biotechnology) for 2 h at 4 o C. Immunoprecipitates were collected on 50 μl Protein G/A Agarose beads by overnight rotation at 4 o C, washed with lysis buffer and resuspended in 50 μl 2X Laemmli buffer.

Techniques: Flow Cytometry, Labeling, Transfection, Fluorescence, Expressing

Journal: bioRxiv

Article Title: N-Cadherin Provides a Cis and Trans Ligand for Astrotactin that Functions in Glial-Guided Neuronal Migration

doi: 10.1101/357541

Figure Lengend Snippet: Drosophila S2 cell adhesion assays were prepared in four conditions: Cdh2;GFP + mCherry;GFP (A) , Cdh2;GFP + Astn1;mCherry (B) , Cdh2;GFP + Astn1;mCherry + ASTN1 Fab (C) , and Cdh2-A390;GFP + Astn1;mCherry (D) . ASTN1-positive cells were adhering to the CDH2-expressing aggregates after 30 min (arrows in B1 ), with more co-aggregation seen after 1 h (arrows in B2 ) and 2 h (arrows in B3 ), indicating heterophilic trans interactions. Significantly lower proportions of cells were adhering to the aggregates in the conditions with cells expressing control vector (A) or Astn1;mCherry blocked with ASTN1 Fab fragments (C) . The proportion of mCherry expressing cells in the CDH2;GFP-positive aggregates is quantified in (E) . Expression of CDH2∆390;GFP did not result in cell aggregation within 2 h (D) , demonstrating the importance of the cadherin ectodomain for homophilic and heterophilic interactions and cell adhesion. ** P < 0.01; *** P < 0.001. Scale bar represents 20 μm in (A - D) and 10 μm in inset in (B3).

Article Snippet: After removing the beads, the lysates were incubated with 3 μg of a rabbit GFP antibody (Invitrogen), rabbit Astn1 antibody, or normal rabbit IgG (Santa Cruz Biotechnology) for 2 h at 4 o C. Immunoprecipitates were collected on 50 μl Protein G/A Agarose beads by overnight rotation at 4 o C, washed with lysis buffer and resuspended in 50 μl 2X Laemmli buffer.

Techniques: Expressing, Plasmid Preparation