Supplemental Data

Supplemental Data

Files in this Data Supplement:

• Supplemental Figure 1 - Consequences of the Grb14 R385N substitution on Grb14-IR interaction measured by in vitro BRET.
  For in vitro BRET experiments HEK293T cells were transfected separately with IR-luc, wild-type pEYFP-N1-Grb14 or mutated pEYFP-N1-Grb14 using FuGENE 6 (Roche). Forty-eight hours later, cells transfected with IR-luc were treated or not with 100 nM insulin for 5 min. Cells were lysed with 150 μl lysis buffer per well (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 10 mM β-glycerophosphate, 0.4 mM Na³VO⁴, 50 mM NaF, 0.8 mM AEBSF, 1 μg/ml pepstatin A, 1 μg/ml leupeptin, 1 μg/ml aprotinin, 1 μg/ml antipain) and centrifuged at 15000g for 15 min at 4^°C. One microliter of the cell extract containing IR-luc was incubated in PBS with 1 microliter of the cell extract containing EYFP-Grb14 (WT or mutant) for 30 min at room temperature. 5 μM coelenterazine h (Interchim) was then added and light emission acquisition at 480 nm and 530 nm was started immediately. A: A representative experiment is shown. B : Quantification of insulin stimulated BRET at time 20 min. Histograms represent the mean ± SD of two independent experiments, expressed as percent of the WT.
• Supplemental Figure 2 - Consequences of the Grb14 S370A and S370D substitutions on Grb14-IR interaction measured by in vitro BRET.
  The experiments were performed as described in supplementary figure 1. A: A representative experiment is shown. B : Quantification of insulin stimulated BRET at time 20 min. Histograms represent the mean ± SD of two independent experiments, expressed as percent of the WT.