Fig. 3

LPI does not induce GPR55-K80A internalisation. a Quantification of ssGPR55 internalisation in transfected HeLa cells by FACS analysis, after live-cell immunostaining using the murine anti-HA antibody (see “Materials and methods” section for details). Cells were stimulated or not with 10 µM soybean LPI and analysed over time. The mean fluorescences obtained are expressed as percentages of the unstimulated sample (unst.), and are indicative of the residual GPR55 plasma-membrane localisation. The efficiency of transfection in these experiments was 55%, and the mean fluorescence of ssGPR55-expressing unstimulated HeLa cells was 3305 ± 397 a.u.. Data are means ± SEM of three independent experiments. b HeLa cells expressing ssGPR55 wild-type and mutants (as indicated) were stimulated for 15 min without or with 10 µM soybean LPI, and then cell-surface localisation of the receptors was quantified by FACS. Data are means ± SEM of three independent experiments, and are mean fluorescences of each sample as percentage of correspondent unstimulated cells. The efficiency of transfection in these experiments was 44%, 42%, 42%, and the mean fluorescences were 1982 ± 247, 1747 ± 325, 1889 ± 443, for ssGPR55 wild-type, ssGPR55-K80A and ssGPR55-Q87A, respectively. *p < 0.05; ***p < 0.005 (Student’s t-tests) versus corresponding unstimulated cells