Fig. 2: KP1 competes with TGF-β1 for TβR2 binding.
From: A Klotho-derived peptide protects against kidney fibrosis by targeting TGF-β signaling
a, b Co-immunoprecipitation (Co-IP) demonstrated that KP1 bound to TβR2. NRK-49F cell lysates (500 µg) and FITC-KP1 (10 µg) were immunoprecipitated (IP) with the anti-TβR2 antibody at 4 °C overnight, followed by immunoblotted (IB) for FITC and TβR2, respectively (a). In the reciprocal experiment, mixtures of cell lysates and FITC-KP1 were immunoprecipitated with anti-FITC, followed by immunoblotted for TβR2 and FITC (b). c The affinity of TβR2 to KP1 was shown. SPR analyses showed concentration-dependent (0.78–25 μM) binding of KP1 to TβR2 immobilized on a sensor chip. A representative sensorgram of two independent experiments is presented, in which the curves (color) with an overlay of the fitting (black) are shown. The fitted constants are ka = 407.7 M−1 S−1; kd = 5.8 × 10−4 S−1; KD = 1.4 × 10−6 M. d The affinity of TβR2 to KP12 was shown. SPR analyses showed concentration-dependent (1.56–100 μM) binding of KP12 to TβR2 immobilized on a sensor chip. A representative sensorgram of two independent experiments is presented, in which the curves (color) with an overlay of the fitting (black) are shown. The fitted constants are ka = 159.5 M−1 S−1; kd = 2.3 × 10−3 S−1; KD = 14.6 × 10−6 M. e The specific binding of FITC-KP1 to TβR2 coated on a microplate. The fluorescence intensity of FITC-KP1 (arbitrary units) was assessed after incubating on a microplate coated with TβR2 protein. P values (from left to right): <0.001, <0.001, and <0.001 (one-way ANOVA with Fisher’s LSD post hoc test). (n = 4 biologically independent cells). Data are presented as mean values ± SEM. f KP1 inhibited the interaction between TβR2 and TGF-β1 dose-dependently. NRK-49F cells were pre-incubated with different amounts of KP1 for 1 h, and then treated with TGF-β1 (2 ng/ml) for 5 min. Cells were collected and immunoprecipitated with anti-TβR2 or anti-IgG. g Negative peptide KP12 did not block the binding of TβR2 and TGF-β1. h KP1 inhibited the binding of TβR1 and TβR2. i KP1 inhibited the interaction of TβR2 and sKlotho. Cells were transfected with sKlotho plasmid for 24 h and incubated with TGF-β1 in the absence or presence of KP1. Cells were collected and immunoprecipitated with anti-TβR2. Source data are provided as a Source Data file.
