Insulin receptor trafficking and interactions in muscle cells - Supplemental Materials

Supplementary Figures and Videos Figure S1. Insulin signalling of C2C12 myoblast overexpressing inter-domain-tagged INSR-A-EGFP. (A) Representative western blot image of C2C12 cells after 5 min of insulin stimulation. Control lanes were samples without overexpressing tagged INSR. # indicates unspecific band, which is previously reported in C2C12 cells (67). It does not always appear (67); in our hands, it didn’t appear in INSR-A-EGFP lanes or other samples in Fig. 1B. A lot more proteins were unfortunately loaded for control samples, shown by α-tubulin bands as loading control. Total AKT and ERK were not successfully blotted due to technical issues. (B) Percentage overexpression of INSR-A-EGFP, showing a moderate overexpression of ~35%. It is calculated by the intensity of the INSRβ-EGFP band divided by the endogenous INSRβ of the same lane. (n = 2 biological replicates.) (C, D) pAKT and pERK normalized by (C) tubulin or (D) INSR (INSRβ band or the sum of INSRβ and INSRβ-EGFP bands). Due to unequal loading, the intensities of WT and INSR-A-EGFP groups are less directly comparable (as the chemiluminescence of western blot is only semi-quantitative with a limited linear range); therefore, each group is normalized by the mean of its own 0 nM insulin condition to make the signalling more comparable. Overall, the pAKT and pERK activations in cells overexpressing INSR-A-EGFP were similar to or no less than the controls. (n = 2 biological replicates.) Figure S2. Potential interaction sites based on per-residue scores along the interactor (target) proteins against the INSR cytoplasmic region (residues 968 to 1372). minD (more accurate) and min-iPAE scores detect interaction sites. pLDDT shows whether the region is structured or unstructured. Shades show confidence errors of 99%. Video 1. Live-cell imaging of surface-labeled INSR-A-SNAP from 0 to 15 min. Video 2. Live-cell imaging of surface-labeled INSR-A-SNAP from 20 to 30 min.