Anti phospho igf 1rβ tyr1135 1136 irβ tyr1150 1151

Cell stimulation and detection of receptor phosphorylation were performed as described previously (33 (link)), using mouse fibroblasts (IR-A, IR-B, and R⁺³⁹). For details, see supporting information. The cells were stimulated with 10 nm concentrations of the ligands for 10 min. Proteins were routinely analyzed using immunoblotting. The membranes were probed with anti-phospho-IGF-1Rβ (Tyr^1135/1136)/IRβ (Tyr^1150/1151) (Cell Signaling Technology). Each experiment was repeated four times. The data were expressed as the contribution of phosphorylation relative to the human insulin (IR-A, IR-B) or IGF-1 (IGF-1R) signal in the same experiment. Mean ± S.D. (n ≥ 4) values were calculated. The significance of the changes in stimulation of phosphorylation in relation to insulin was calculated, using one-way analysis of variance with Dunnett's test comparing all analogs versus control (i.e. insulin).
Ligand–dose response IGF-1R autophosphorylation levels for selected analogs were determined, using the In-Cell Western assay adapted for chemiluminescence as described (34 (link)). Data were subtracted from background values and expressed as the contribution of phosphorylation relative to the 10 nm IGF-1 signal. Experiments were repeated at least four times. Log(agonist) versus response curve fitting of data was carried out with GraphPad Prism 5.

Cell stimulation and detection of receptor phosphorylation were performed as described previously [44 (link)], using mouse fibroblasts transfected with human IR-A or human IGF1R. Briefly, the cells were stimulated with 10 nM concentrations of the ligands for 10 min. Proteins were routinely analyzed using immunoblotting. The membranes were probed with anti-phospho-IGF-1Rβ (Tyr1135/1136)/IRβ (Tyr1150/1151) (Cell Signaling Technology). Each experiment was repeated four times. The data were expressed as the contribution of phosphorylation relative to the human insulin (IR-A) or IGF-1 (IGF1R) signal in the same experiment. Mean ± S.D. (n≥4) values were calculated. The significance of the changes in stimulation of phosphorylation in relation to IGF2 was calculated, using one-way analysis of variance (ANOVA), with Dunnett’s test comparing all analogs versus control i.e. IGF2 [44 (link)].

The abilities of analogs to induce autophosphorylation of IGF-1R in membranes of mouse fibroblast transfected with human IGF-1R and with deleted mouse IGF-1R were determined, as described by Macháčková et al. (Machackova et al. 2019 (link)). Briefly, the cells were stimulated in 24-well plates (Schoeller) (4 × 104 cells per well) after 4 h of starving in serum-free medium. The cells were stimulated with 10 nM concentration of the ligands for 10 min. Stimulation was stopped by snap-freezing. Proteins were routinely analyzed, using immunoblotting and horseradish peroxidase-labeled secondary antibodies (Sigma-Aldrich). The membranes were probed with antiphospho-IGF-1Rβ (Tyr-1135/1136)/IRβ (Tyr-1150/1151) (Cell Signaling Technology). The blots were developed, using the SuperSignal West Femto maximum sensitivity substrate (Pierce), and analyzed using the ChemiDoc MP imaging system (Bio-Rad). The autophosphorylation signal density generated by each ligand on Western blotting was expressed as the contribution of phosphorylation relatively to the IGF-1 (IGF-1R fibroblasts) in the same experiment. Means were calculated from two to four independent experiments (n = 2–4), depending on the available amount of material, and were compared with native IGF-1.