Fig. 2

ssGPR55-K80A and ssGPR55-Q87A mutants have impaired LPI-induced stimulation of MAPKs. a FACS analysis with an anti-HA antibody of shGPR55-HeLa clones transfected with equal amounts (2.5 µg/well, in six-well-plates) of empty vector (pcDNA3.1), or of vector coding for ssGPR55 wild-type (ssGPR55) or the mutants (as indicated). The median fluorescence intensities and (% coefficient of variation) for these gated GPR55-transfected cells were: 1134 (132.5) for ssGPR55; 1286 (139.8) for ssGPR55-K80A; 1164 (134.8) for ssGPR55-Q87A. b Twenty-four hours after transfection, shGPR55-HeLa clones were serum deprived for 2 h, then stimulated with 10 µM 18:0 LPI for the indicated times. Western blotting of phosphorylated (p-ERK1/2, p-p38) and total ERK1/2 and p38 are shown from a representative experiment. c Densitometric analysis by arbitrary units (a.u.) of ERK2 (top) and p38 (bottom) phosphorylation levels, normalised for the correspondent protein levels. Data are expressed as fold of unstimulated ssGPR55-overexpressing cells (unst. ssGPR55), and are means ± SE of three independent experiments. *p < 0.05, (Student’s t-tests) versus corresponding unstimulated cells