Fig. 1. GPR37 is expressed at the cell surface of COS-7 cells and internalises after HA treatment. (A-I) COS-7 cells were transfected with GPR37 with (B,D-I) or without (A,C) FLAG tag, immunostained with anti-GPR37 antibody (A,C,D) or with anti-FLAG antibody (B,E-I) and visualised with alkaline phosphatase-coupled secondary antibodies for light microscopy (A) or with Cy3-coupled antibodies for confocal analysis (B-I). Cells were permeabilised (perm) by fixation with 1% acetic acid in ethanol and by washing with Triton X-100, except in D, where living cells were incubated with the primary antibody before fixation (non-perm) to show surface staining. (E-I) COS-7 cells 48 hours after transfection with GPR37-FLAG were treated at 37°C with 2 nM HA for 0, 10, 20, 30 and 60 minutes, respectively, and immunostained with anti-FLAG antibody.