Figure S2.

Teneurin, Latrophilin, and FLRT Interaction Studies, Related to Figure 2

(A) Teneurin and Latrophilin constructs were expressed in HEK293 cells with an intracellular mVenus and extracellular HA or Myc tag, respectively. We visualized cell surface expression with anti-HA or anti-Myc staining of fixed non-permeabilised cells. The staining shows that the constructs used in this study were all successfully expressed at the cell surface. Scale bar = 150 μm. (B) We tested the binding of Lphn1 (Lec-Olf) wild-type, single or double mutant proteins, clustered with anti-His and anti-mouse Alexa-594 (red), to HEK293 cells expressing Ten2 (green). Lphn1 and the non-FLRT binding (FL) mutant bind to Ten2. Non-Teneurin binding (TL) mutants do not bind. (C-E) SPR response curves are shown. The response units (y axis) are plotted against the time in seconds (x axis). (C) We immobilised 700 response units of murine Lphn1 (ecto), Lphn2 (ecto), or Lphn3 (ecto) on separate flow cells and injected a concentration series of mouse Teneurin 2 protein (highest concentration = 3.65 μM). (D, E) We immobilised 440, 300 and 1200 response units of FLRT2 (ecto) or FLRT3 (ecto). We injected Teneurin and Lphn proteins using the same concentration series in each experiment (highest concentration = 660 nM). (F) mVenus-tagged Ten2 was pulled down from HEK293T-cells co-transfected with FLAG-FLRT2, after incubating with either Lphn3-transfected cells or untransfected control cells. Anti-FLAG western blots show that FLRT2 is preferentially pulled down in the presence of Lphn3-expressing cells, compared to untransfected controls. Three representative repeats are shown. FLRT is highlighted by the arrow head. (G) Quantification of results shown in F. Results averaged from 6 experiments. Statistical significance was determined with a two-tailed unpaired t test where ^∗∗∗p = 0.0003. Error bars show the s.e.m.