Fig. 3. BRL1 and BRL3 are membrane-localized receptors that bind BL. (A) Specific [³H] –BL binding for immunoprecipitated membrane fractions of BRL1 and (B) BRL3. Shown are the Kd values from one representative experiment of three replicates. BRI1-GFP was used as a positive control in our experiments (Kd=55.5 nM±0.08). (C) Western blot developed with anti-GFP antibodies showing the amount of BRI1::GFP, BRL1::GFP, BRL2::GFP and BRL3::GFP fusion proteins in the immunoprecipitated membrane fractions used for the [³H] –BL binding assays. (D) Subcellular localization of the BRL receptors. Confocal images of hypocotyl epidermal cells of light-grown 7-day-old seedlings carrying 35S::BRL1-GFP and 35S::BRL3-GFP fusion constructs show that both proteins are localized to the plasma membrane. Scale bar: 25 µm. (E) The specific [³H] –BL binding activity for each BRL protein and BRI1. BRL2 does not bind BL specifically. In these graphs, BRL1 binding appears lower than BRL3 or BRI1 due to the level of protein expressed in the transgenic lines used for this experiment.