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Figure 2


Fig. 2. Inducible, stable expression of GPR37 prevents aggregate formation. (A) CHO-K1, COS-7 and HEK-293 cells were transiently transfected with GPR37, and membrane fractions were assayed by immunoblotting (IB) with anti-GPR37 antibody ({alpha}-GPR37). (B) COS-7 cells transiently transfected with GPR37-FLAG were cell-surface biotinylated, and the solubilised membrane fraction was immunoprecipated (IP) with anti-FLAG antibody ({alpha}-FLAG) and visualised after immunoblotting with avidin. (C) GPR37 was introduced stably into the flip-in cell line HEK-T-REx, where GPR37 expression is inducible by doxycycline (DOX). Membrane fractions were subjected to western blotting with anti-GPR37 antibody ({alpha}-GPR37) with (first lane) and without (second lane) induction for 24 hours with doxycycline. (D-I) HEK-T-REx-GPR37 cells with (D,F,H,I) and without (E,G) doxycycline induction for 24 hours were immunostained with anti-GPR37(R2) antibody after permeabilisation (D,E) and with anti-GPR37 antibody without permeabilisation (F-I). (H,I) HEK-T-REx-GPR37 cells were treated with 2 nM HA for 0 and 10 minutes at 37°C, respectively, fixed with 2% formaldehyde for 10 minutes and subsequently immunostained with anti-GPR37 antibody.





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