Figure 3

Chimeras characterization. A. HeLa cells expressing Mut-GFP (A), Mut (B), IR-B-SCFP (C) or IR-B (D) were stimulated with 100 nM rhIns for 10 min and fixed. Immunofluorescence assays were performed with anti-phospho-IR and a secondary antibody conjugated with Alexa fluor 555. Cells expressing Mut were labeled with 0.2 μM ACP-S and 1 μM CoA-488 before stimulation. E. Cells expressing wt IR-B or the mutant were stimulated with 100 nM rhIns for 5 min and lysates were analyzed by Western blot using anti-pY20 and anti-IR-β subunit. Quantification was performed by densitometry. The ratio pY20 / IR was measured for each lane and fold induction was calculated with respect to basal levels. Results are expressed as the mean ± s.e.m. (*: p=0.002; n≥3). ´p´ means phospho-antibodies.