Fig. 2: Ras-LOCKR-S can report subcellular endogenous Ras activity in multiple cell types.

a, Schematic of subcellularly targeted Ras-LOCKR-S, EGF receptor (EGFR) and CIAR at the PM (CIAR-PM). Localization sequences: PM: N-terminus of Lyn; Golgi: N-terminus of eNOS. b, Top, representative images of 293T cells transfected with localized Ras-LOCKR-S (Key with localization sequence, Cage untargeted) and stained for localization markers. Bottom, line trace comparison of CFP signal from Ras-LOCKR-S Key and localization marker. Scale bar, 5 μm. c,d, CIAR-PM-293 cells expressing Ras-LOCKR-S WT or NC localized to PM (c) or Golgi (d) were stimulated with either 100 ng ml⁻¹ EGF or 250 nM A115. Left, representative epifluorescence images using ×60 objective (CFP channel from Key, FRET channel and pseudocolored raw FRET ratio) from three biologically independent experiments. Right, normalized FRET ratio changes (n = 16 cells per condition for WT PM and NC Golgi experiments, n = 20 cells per condition for NC PM experiments and n = 15 cells per condition for WT Golgi experiments). Scale bars, 10 μm. e, Average normalized FRET ratio responses of Jurkat T cells expressing Ras-LOCKR-S localized to Golgi or PM stimulated with either 5 μg ml⁻¹ of α-CD3ε + α-CD28 (left) or 5 μg ml⁻¹ of α-CD3ε + intracellular adhesion molecule 1 (ICAM-1; right) coated on the plate (n = at least 10 cells per condition). Solid lines in c,d indicate a representative average timecourse from three biologically independent experiments, with error bars representing s.e.m. Solid lines in e indicate average timecourses of FRET ratio changes from all cells combined from three experiments, with error bars representing s.e.m. WT, wild-type, NC, negative control.