Figure 5

Dimers wild type / mutant at the plasma membrane and signaling. A. HeLa cells expressing Mut, IR-B-SCFP or both were treated with 2 μM ACP-S and 5 μM CoA-biotin for 30 min. Lysates were incubated with SA-agarose beads for 1 h at 4°C. Precipitates and total fractions were analyzed by Western blot with anti-IR-β subunit. B. HeLa cells were co-transfected with 0.1 μg pcDNA3-IR-B and different amounts of the mutant or EV, stimulated with 100 nM rhIns for 5 min and assayed by Western blot. Quantification was performed by densitometry measuring pY20 signal with respect to the first lane (basal) (*: p<0.05, n=4). C. HeLa cells co-expressing Mut and IR-B were labeled with 2 μM ACP-S and 2 μM CoA-488 and then incubated with 50 nM BAC-Ins and 1 nM QD655. Cells were directly fixed or incubated for 30 min at 37°C and then fixed or acid treated before fixation. Samples were imaged by confocal microscopy. Scale bars: 10 μm. D. Internalization analysis. Results are expressed as the mean ± s.e.m (p<0.005; n=6-32 cells).