Fig. 2: AdipoR2 is a direct binding target of SCM-198.
a Cell activity analysis of AML12 cells treated with different amounts of SCM-198-Biotin, with CCK8 assay. b Cell activity analysis of AML12 cells treated with SCM198 or SCM-198-Biotin, after H2O2 exposure, showing that SCM-198-Biotin had a similar effect to SCM-198. c Fluorescence image of mouse AML12 hepatocytes after incubation with SCM-198-FITC (green) at 37 °C for 24 h. The nucleus was stained with DAPI, and the cell membrane was stained with WGA (red). Scale bars, 10 μm. d Schematic illustration of the affinity-purification approach for identifying SCM-198 targeting proteins from cytomembrane extracts of AML12 mouse hepatocytes incubated with SCM-198-Biotin probe. Created in BioRender. Lin, G. (2024) https://BioRender.com/s19x956. e Immunofluorescence staining of AdipoR2 on liver sections of TAA/APAP- intoxicated mice, 24 and 48 hpi. Mice were given saline or SCM-198 at 24 hpi. Control: uninjured. Scale bar: 50 µm. f Immunoblotting of AdipoR2 and AdipoR1 in eluate obtained from AML12 hepatocytes, following the procedure shown in (d). Biotin, negative control; Non-labeled SCM-198, a competitor for SCM-198-Biotin. Similar results were obtained from 3 independent repeats. g Immunoblotting of AdipoR2 in TAA/APAP- intoxicated WT and Adipor2-/- mouse liver tissue extracts, affinity purified with SCM-198-Biotin probes. β-actin was used as the input loading control. h Differential scan fluorimetry analysis showing the changes in the enthalpy of recombinant human ADIPOR2 incubated with SCM-198 in PBS buffer at indicated temperatures. Tm: thermal transition midpoint. i Microscale thermophoresis analysis of the binding affinity between the ADIPOR2 and SCM-198. The fitted Kd value for ADIPOR2-SCM-198 interaction is shown. Error bars indicate the SEM of three separate experiments. ADIPOR2(180-386) was used as control. j SPR binding curves and equilibrium fits for the interaction of ADIPOR2 with SCM-198 (at different concentrations). Data in (a, b) are presented as mean ± SEM. n = 3 experimental repeats. p-value was determined using SPSS v20 for t test, two-tail analysis. n.s., not significant. Similar results were obtained in 3 independent repeats (c, e, g). Source data are provided as a Source Data file.
