Co-immunoprecipitation and overlay assays indicate interaction of 14-3-3 and IRS-2 upon IGF-1/insulin stimulation.

<p><i>A.</i> HEK293 cells were transiently transfected with GFP or GFP-IRS2 and after serum starvation cells were incubated with 50 ng/ml IGF-1 for 30 min or after preincubation with 100 nM wortmannin for 30 min. 400 µg total protein was used for immunoprecipitation with 14-3-3 antibody (C-17) and samples were separated on 5–15% gradient gel. Upper membrane was incubated with IRS-2 antibody, lower membrane with 14-3-3 antibody (K-19). <i>B.</i> HEK293 cells were transfected with GFP-IRS2 and stimulation was carried out after starvation for serum overnight with 50 ng/ml IGF-1 for 30 min or subsequently after preincubation with 1 µM PI-103 for 30 min. 250 µg of total protein was pulled down using GFP-Trap®. SDS-PAGE followed transfer onto nitrocellulose membranes. Overlay assay followed stripping of the membrane and reprobing with GFP antibody as loading control. <i>C.</i> Extent of interaction was quantified by scanning densitometry of blots and normalization for GFP-IRS2 serum starved condition (mean ± SEM; n = 4; *p<0.05 serum starved vs. IGF-1 or IGF-1 vs. PI-103/IGF-1). <i>D</i>. Male C57Bl/6 mice were fasted overnight and injected intravenously with 2 IU (international units) insulin. After 10 min liver was taken and 500 µg of total protein was immunoprecipitated with IRS-2 antibody. After performing overlay assay membrane was stripped and reprobed with IRS-2 antibody as loading control. Two mice of each group are shown. <i>E</i>. Densitometric analyses of 14-3-3 interaction with IRS-2. Overlay signal was normalized for total IRS-2 protein content (mean ± SEM; n = 4; *p<0.05 fasted vs. insulin). <i>F</i>. Male C57Bl/6 mice were fasted overnight, refed for 4 hours or injected intraperitoneally with insulin for 30 min. Procedure as in <i>G</i>. Four mice of each group are shown. <i>G</i>. 14-3-3 interaction with IRS-2 was quantified by scanning densitometry of immunoblots and normalization for IRS-2 protein (mean ± SEM; n = 4; *p<0.05 fasted vs. refed and insulin stimulation).