Figure 6

Mutant IR blocks insulin induced AP-1 activity without affecting Akt activation. A. HeLa or HEK293 cells co-expressing AP1-Luc, IR-B and different amounts of the mutant were starved for 24 h and then stimulated with 100 nM rhIns for 16 h. Luciferase activity was measured and fold induction was calculated (*: p≤0.03, **: p=0.003; n≥3). B. Akt translocation to the plasma membrane: HeLa cells co-expressing IR-B-SCFP and Akt-HA were starved overnight and then stimulated with 100 nM rhIns for 5 min. After fixation samples were stained with anti-Akt and a secondary antibody conjugated with Alexa fluor 555. Images were acquired by confocal microscopy and quantification of Akt re-distribution was evidenced (*: p<10^-6; n=34-40 cells). Scale bars: 10 μm. C. Similar experiment was performed using anti-phospho-Akt in cells co-expressing Akt-HA with: i) IR-B-SCFP, ii) Mut iii) both (n=15-23 cells). D. Cells co-transfected with 0.1 μg pcDNA3-IR-B and different amounts of the mutant or EV were stimulated with 100 nM rhIns for 5 min and assayed by Western blot. Quantification was performed by densitometry measuring phospho-Akt signal normalized to the first lane (basal) (*: p<0.05, n≥3). ´p´ means phospho-antibodies. Results are expressed as the mean ± s.e.m.