IR activation by IGF-II and IGF-II-biotin. A. HeLa cells over-expressing IR-B were starved for 16 hours and stimulated for 5 min with 100 nM rhIns, BAC-Ins, IGF-II or IGF-II-biot at 37°C. Lysates were analyzed by Western blot with anti-phosphorylated-Tyrosine (p-Tyr) and anti-IR-β subunit antibodies; and anti-phosphorylated-ERK 1/2 (p-ERK) and anti-ERK 1/2 antibodies. B. HeLa cells were transfected with pcDNA3-IR-B or EV stimulated or not with insulin for 5 min (bands were cut from the same gel). C. HeLa cells expressing IR-B-SCFP stimulated with 100 nM IGF-II, IGF-II-biot or rhIns for 5 min at 37°C, fixed in cold methanol and analyzed by immunofluorescence with anti-phosphorylated-IR-β subunit (Tyrosine 1361) antibody and a secondary antibody conjugated with Alexa fluor 555. Imaging was performed by confocal microscopy (Olympus Fluoview FV1000). Scale bars: 10 μm. Arrows indicate that non transfected cells do not show activation signal upon ligand stimulation. Arrows indicate non transfected cells. D. Colocalization analysis performed with Image J. Product of the differences from the mean (PDM) plots and Manders coefficients for each channel (MpIR-555 and MSCFP) are shown for stimulation with IGF-II and rhIns.