Additional file 8 of Peptide targeting of lysophosphatidylinositol-sensing GPR55 for osteoclastogenesis tuning

Additional file 7. Figure S5. Peptide-P1 specifically binds to murine GPR55 in RAW264.7 cells. (a) Time course of binding of 40 µg/mL (26.8 µM) FITC-conjugated Peptide-P1 (FITC-P1) or the scrambled (KCLTSNCPK) peptide (FITC-Scr) to RAW264.7 cells at 37 °C. Peptide binding evaluated in subsequent FACS analysis of cell-associated FITC-fluorescence is shown, quantified as mean fluorescence increase compared to cells incubated in the absence of any peptide, with data representative of three independent experiments. The extrapolated apparent Kd for FITC-P1 was 22.7 µM. (b) Peptide specificity towards GPR55 was determined by incubation of 40 µg/mL FITC-labelled peptides with RAW264.7 cells treated with non-targeting (si-NT+siGLO) or Gpr55-targeting (si-GPR55+siGLO) siRNAs for 15 min at 37 °C. Peptide binding was subsequently evaluated by FACS analysis of cell-associated FITC fluorescence in the siGLO-positive cells, quantified as mean fluorescence increase compared to cells incubated in the absence of any peptide (see Methods). Data are means ±SEM of four independent experiments. *p <0.05 (Student’s t-test).