Fig. 10: FGF2-GFP secretion is modulated by PI(4,5)P2 levels at the plasma membrane surface.
CHO-K1 cells were induced to express FGF2-GFP overnight by 1 µg/mL doxycycline. A Representative confocal images of mock-treated and PI(4,5)P2-treated CHO-K1 cells. Cells were fixed either at 0, 20, 40, and 60 min after disruption of plasma membrane PI(4,5)P2 asymmetry. Fixed cells were stained with high affinity PI(4,5)P2 binding protein PHX2-Halo (40 nM). The Halo tag was visualized using Halo ligand Alexa 660 (75 nM). B Quantification and statistical analysis of surface FGF2-GFP levels for individual cells that were left untreated, mock-treated, and PI(4,5)P2-treated. Cells were fixed at 0, 20, 30, 40, and 60 min after the heparin wash. Cells were fixed using 3%PFA in PBS for 13 min at room temperature. Surface FGF2-GFP was detected using rabbit polyclonal anti-GFP-Alexa647 antibody at 1:400 dilution. Each light grey dot represents a single cell coming from 3 to 9 independent experiments. Mean values from different replicates are presented in the solid colors. Unspecific background (BG) from antiGFP-AlexaFluor-647 signal after heparin wash for each condition was subtracted. For statistical test, One-way ANOVA was performed on mean values from 3 replicas in Prism. Not significant (ns) P > 0.5, ** P < 0.01. Data distribution was assumed to be normal. Source data are provided as a Source Data file.
