Mouse Anti-INSR Recombinant Antibody (clone 83-7) (CAT#: PABL-246)

Figure 1 Immunohistochemistry (1µg/ml) staining insulin receptor alpha in human pancreas using an automated system (DAKO Autostainer Plus).

Figure 2 Overlay histogram showing Jurkat cells stained with 83-7 (red line).

The cells were fixed with 4% paraformaldehyde (10 min) and incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions. The cells were then incubated with the antibody for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG1 used under the same conditions. Acquisition of>5,000 events was performed. This antibody gave a positive signal in Jurkat cells fixed with methanol (5 min) used under the same conditions.

Figure 3 (D-G) Cell surface expression of INSR mutants as determined by flow cytometry.

Stacked overlay single parameter histograms displaying intensity of INSR-FITC fluorescence on the X-axis and number of events on the Y-axis. Isotype control IgG (light grey) was used as a negative control to generate a negative gate to determine the percentage of the population positive for anti-INSR antibody binding (numbers). Rightward shift of the peak (blue 83-7, dark cyan 83-14, orange 18-44, purple 18-146) from the IgG control is a function of both mutant INSR expression and antibody affinity.

Brierley, G., Siddle, K., & Semple, R. K. (2017). Evaluation of anti insulin receptor antibodies as potential novel therapies for human insulin receptoropathy. BioRxiv, 223883.

Figure 4 Insulin- and antibody-stimulated autophosphorylation of WT and mutant INSR.

CHO FlpIn cells stably expressing either human WT or mutant INSR (as indicated) were serum starved prior to 10 minute stimulation with increasing concentrations of either insulin, antibody (83- 7, 83-14, 18-44, 18-146 or control IgG, solid lines) or increasing concentrations of insulin in the presence of 10nM antibody (broken lines). Cells were lysed and myc-tagged receptors were immunocaptured on 96-well plates and then incubated with biotin-conjugated 4G10 platinum antibody to detect phosphorylated tyrosine residues. Europium-labelled strepavidin was used to detect bound anti-phosphotyrosine antibody 4G10 by time-resolved fluorescence.

Brierley, G., Siddle, K., & Semple, R. K. (2017). Evaluation of anti insulin receptor antibodies as potential novel therapies for human insulin receptoropathy. BioRxiv, 223883.

Figure 5 Insulin and antibody stimulated glucose uptake via WT and mutant INSR.

3T3-L1 MmINSRKD hINSR (mutant as indicated) adipocytes were grown in the presence of 1μg/ml DOX for 10 days prior to overnight serum-starvation on day 15 of differentiation. The cells were stimulated for 30 minutes with either 10nM insulin, 10nM antibody (83-7, 83-14, 18-44, 18-146, or control IgG) or 10nM insulin containing 10nM antibody prior to the addition of 2-Deoxy-D-glucose for 5 minutes. Cells were then washed, lysed and assessed for 2-Deoxy-D-glucose uptake. (A–I) Glucose uptake stimulated by treatment with antibodies 83-7 and 83-14.

Brierley, G., Siddle, K., & Semple, R. K. (2017). Evaluation of anti insulin receptor antibodies as potential novel therapies for human insulin receptoropathy. BioRxiv, 223883.