Figure 4. GM3 depletion activates IR/IGF-1R and downstream signaling.

After starvation, KCs were treated with vehicle (Ctrl), insulin, or IGF-1 with (right) or without (left) supplemental glucose (G). Phosphorylation of IR (a) and IGF-1R (b) in whole cell lysates (top rows) was detected by p-IR and p-IGF-1R antibodies. (c) Association of immunoprecipitated IR and IGF-1R with IRS-1 (middle rows) and each other (bottom rows). First lane shows immunoprecipitation with IgG alone. (d) IRS-1 detected with anti-IRS-1 antibody (top row); phosphorylation of immunoprecipitated IRS-1 with PY-20 antibody (bottom row). First lane shows immunoprecipitation with IgG alone. Graph: results of 4 blots from 4 different experiments. (e) AKT (top) and p-AKT Ser⁴⁷³ (middle) and blots representative of 3 experiments. Blots in 4a–d are representative of 4 separate experiments; the blot in 4e of 3 separate experiments. Graphs for 4a, b, and d show data as quantified densitometrically and expressed as means±S.D. To be able to compare cells treated with and without excess glucose, all experiments were run in parallel. Results were normalized to GAPDH expression and then phosphorylation was expressed in comparison with WT KCs treated with the primary ligand of the receptor of interest (i.e., insulin for IR and IGF-1 for IGF-1R), which was assigned a fold change of 1. To show statistical significance, asterisks (*) compare GM3S^−/− vs. WT in all graphs with **p<0.01, ***p<0.001; carets (^) compare with vs. without supplemental glucose in all graphs for GM3S^−/− and WT KCs, ^^^p<0.001.