Insulin

Direct ELISA assays were performed similarly to those reported previously. Briefly, high-binding Costar 96-well plates (Corning) were coated with 0.5 μg salmon sperm ssDNA (Abcam), calf thymus dsDNA (Sigma-Aldrich), lipopolysaccharide from E. coli (Sigma-Aldrich), chicken egg white lysozyme (Sigma-Aldrich), or 0.25 μg insulin (Fitzgerald) and incubated overnight at 4 °C. The next morning, plates were washed three times using wash buffer (PBS pH 7.5, 0.001% Tween), were blocked using blocking buffer (PBS pH 7.5, 0.1% Tween-20, 1 mM EDTA, 2% BSA) for two hours at room temperature, and then were washed three times with wash buffer. Following blocking, nanobodies (200 μL) were incubated at the indicated concentrations at room temperature in PBS pH 7.5 for two hours. After three more washes, plates were incubated with HRP-anti V5 antibody (Abcam ab1325, 1:10,000 dilution) in PBS + 2% BSA for one hour at room temperature. Plates then were washed three times with wash buffer and 1-Step ABTS substrate solution (100 μL, Thermo Scientific) was added to the plates, which were then incubated in the dark for 20 min. Stop solution (1% SDS in PBS, 100 μL) was added to each plate and absorbance at 405 nm was measured using a Spectromax M5 microplate reader. Results were analyzed in GraphPad Prism.

MLE-12 cells were maintained in HITES medium consisting of DMEM/F12 (ATCC, 30-2006), 2% fetal bovine serum (FBS), 0.005 mg/ml insulin, 0.01 mg/ml transferrin (Fitzgerald, 31C-CH1026), 30 nM sodium selenite (Santa Cruz, 253595), 10 nM hydrocortisone (Sigma, H0888), 10 nM β-estradiol, 10 mM HEPES (Gibco, 15630106), 2 mM l-glutamine (Gibco, 25030081), and 1% Pen-Step (Gibco, 15140122). H441 cells were maintained in RPMI-1640 (ATCC, 30-2001) medium supplemented with 10% FBS and 1% Pen-Strep.