Fig. 4: HFt binds to SRCR domain of MSR1.
a Western blot analysis of MSR1 protein expression in HEK293 cells at 24 h after transfection with the control plasmid (p-mCherry2) or the plasmid encoding the MSR1 gene (p-MSR1-mCherry2). Representative western blot images are shown. b and (c) Flow cytometry analysis of cell surface expression of MSR1 in HEK293 cells at 24 h after transfection with the control plasmid (p-mCherry2) or the plasmid encoding the MSR1 gene (p-MSR1-mCherry2). Flow cytometry data are presented as the mean fluorescence intensity (MFI) of BV421. Data are presented as mean ± SEM from n = 3 independent replicates. d Flow cytometry analysis of internalized AcLDL-AF488 (5 μg/ml) and HFt-AF488 (50 μg/ml) within 30 min at 37 °C by HEK293 cells, transfected with either p-mCherry2 or p-MSR1-mCherry2. Flow cytometry data are presented as mean fluorescence intensity (MFI) of fluorescently labeled ligands. Data are presented as mean ± SEM from n = 3 independent replicates. e, f Representative confocal microscopy images of internalized HFt-AF488 (50 μg/ml) (e) and AcLDL-AF488 (5 μg/ml) (f) (green) within 30 min at 37 °C by HEK293 cells, transfected with either p-mCherry2 or p-MSR1-mCherry2 (red). Afterwards, cells were fixed and stained with Hoechst 33342 (blue). Scale bar = 20 μm. g Optimization of AlphaScreen assay conditions by cross-titration of His-MSR1 (0.41–100 nM, 3-fold serial dilution) and Biotin-HFt (0.022–50 nM, 3-fold serial dilution) at fixed 10 μg/ml concentration of both nickel chelated acceptor and streptavidin donor beads. Data shown are from a single pilot run conducted for assay optimization. h Competitive effect of unlabeled HFt on AlphaScreen signal generated by His-MSR1 and Biotin-HFt interaction. Fixed concentrations of 4 nM His-MSR1 and 2 nM Biotin-HFt were used below the hook point of the bead assay (g) in the presence of increasing concentration of unlabeled HFt (0.076–1500 nM, 3-fold serial dilution). IC50 potency of unlabeled HFt in displacing Biotin-HFt from interaction with His-MSR1 was calculated by fitting it to a four-parameter nonlinear regression. Data presented as mean ± SEM from n = 3 independent replicates. i Spectral shift dose–response curve between unlabeled HFt and His-MSR1 conjugated with the RED-tris-NTA dye. Data was analyzed using software provided by the manufacturer and is presented as the emission fluorescence ratio 670/650 vs. log(ligand concentration). Kd value was calculated by fitting data with a 1:1 binding model. Data presented as mean ± SEM from n = 5 independent replicates. j Schematic representation of the full-length MSR1 and SRCR domain deletion mutant of MSR1. Created in BioRender. Taciak, B. (2024) https://BioRender.com/a34q680. k Western blot analysis of MSR1 protein expression in HEK293 cells at 24 h after transfection with the control plasmid (p-mCherry2), the plasmid encoding the full-length MSR1 gene (p-MSR1-mCherry2) or the plasmid encoding SRCR domain deletion mutant of MSR1 gene (p-ΔMSR1-mCherry2). Representative western blot images are shown. l and (m) Flow cytometry analysis of internalized HFt-AF488 (50 μg/ml) within 30 min at 37 °C by HEK293 cells, transfected with the control plasmid (p-mCherry2), the plasmid encoding the full-length MSR1 gene (p-MSR1-mCherry2) or the plasmid encoding SRCR domain deletion mutant of MSR1 gene (p-ΔMSR1-mCherry2). Flow cytometry data are presented as the mean fluorescence intensity (MFI) of HFt-AF488. Data are presented as mean ± SEM from n = 4 independent replicates. n Representative confocal microscopy images of internalized HFt-AF488 (50 μg/ml) within 30 min at 37 °C by HEK293 cells, transfected with the control plasmid (p-mCherry2), the plasmid encoding the full-length MSR1 gene (p-MSR1-mCherry2) or the plasmid encoding SRCR domain deletion mutant of MSR1 gene (p-ΔMSR1-mCherry2) (red). Nuclei were stained with Hoechst 33342 (blue). Scale bar = 20 μm. The two-way ANOVA followed by Sidak’s multiple comparisons post hoc test was used for statistical analysis. For all panels, ****P ≤ 0.0001. Source data are provided as a Source Data file.
