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Figure 3


Fig. 3. HA colocalises with and binds to GPR37. (A-D) Interaction of HA and GPR37 analysed by FRET. Shown is a typical example of FRET between HA and an extracellular epitope of GPR37. COS-7 cells transiently transfected with GPR37 were treated with 2 nM HA for 20 minutes on ice to prevent internalisation, followed by incubation for 20 minute on ice with the antiserum against HA ({alpha}-HA). After fixation with 4% formaldehyde in PBS, cells were immunostained with anti-GPR37 antibody ({alpha}-GPR37). GPR37 immunoreactivity was visualised with Cy3 (A,B) and that of HA with Alexa Fluor 488 (C,D). (A) The Cy3 signal (GPR37) is shown after excitation at 568 nm. (B) A discrete area was photobleached using intense 568 nm laser. The Alexa Fluor 488 signal (HA) after excitation at 488 nm is shown before (C) and after (D) photobleaching. In this example, the Alexa Fluor 488 signal was increased by 18%. (E-G) The neuroblastoma cell line NH15-CA2 was used as a positive control to show specific Cy3B-HA binding to endogenous HA receptors. (E) NH15-CA2 cells endogenously express GPR37, as visualised with anti-GPR37(R1) antibody [{alpha}-GPR37(R1)]. (F,G) Binding is optimal at 150 nM of Cy3B-HA after incubation for 10 minutes at 37°C (Cy3B-HA) and is inhibited by pretreatment for 50 minutes at 37°C with 100 nM unlabelled, monomerised HA (Cy3B-HA comp). (H-J) HEK-T-REx-GPR37 cells bound Cy3B-HA only after GPR37 induction with doxycycline (±DOX), and binding was competed with unlabelled HA (+DOX comp).





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