Journal: bioRxiv

Article Title: GPLD1 functions as a scavenger chaperone mediating lysosomal degradation of extracellular aberrant proteins

doi: 10.1101/2025.07.16.665076

Figure Lengend Snippet: ( A ) Plasma proteins interact with SAA1. Bovine plasma was mixed with recombinant ALFA-SAA1, co-immunoprecipitated with ALFA nanobody beads, and analyzed by Ruby staining. Arrowheads indicate SAA1-binding proteins. ( B ) Schematic of the RFP-GFP internalization assay. Upon internalization, the protein of interest (POI) and GFP are degraded by lysosomal proteases, whereas RFP is resistant to lysosomal degradation and accumulates in lysosomes. ( C ) RFP intensity increases when GPLD1-RFP-GFP is incubated with SAA1. HEK293 cells were treated with serum-free medium containing POI-RFP-GFP with or without SAA1 for 18 h and analyzed by flow cytometry. ( D , E ) GPLD1-RFP-GFP is degraded in lysosomes. HEK293 cells were treated with serum-free medium containing GPLD1-RFP-GFP with or without ALFA-SAA1 in the presence or absence of bafilomycin A 1 (BafA) for 18 h. Cells were analyzed by flow cytometry (D) or immunoblotting using anti-mCherry and anti-β-tubulin antibodies (E). The bar graph shows relative fluorescence intensities normalized to non-treated cells (n = 3). Data are presented as mean ± SEM.

Article Snippet: Rabbit polyclonal antibodies against sfGFP, mCherry, and the ALFA tag were generated by Eurofins using full-length mCherry protein and the ALFA peptide as antigens.

Techniques: Clinical Proteomics, Recombinant, Immunoprecipitation, Staining, Binding Assay, Incubation, Flow Cytometry, Western Blot, Fluorescence

Journal: bioRxiv

Article Title: GPLD1 functions as a scavenger chaperone mediating lysosomal degradation of extracellular aberrant proteins

doi: 10.1101/2025.07.16.665076

Figure Lengend Snippet: ( A ) GPLD1-RFP-GFP internalization is enhanced in the presence of misfolded proteins. GPLD1-RFP-GFP was preincubated with luciferase (42°C for 30 min) or ALFA-ESD (50°C for 1 h) in serum-free medium. HEK293 cells were cultured with medium containing GPLD1-RFP-GFP with or without substrate for 18 h and analyzed by flow cytometry. The bar graph shows relative fluorescence intensities per cell normalized to untreated controls (n = 3). Data are presented as mean ± SEM. ( B ) GPLD1 is transported into lysosomes. HuEhT cells were treated with serum-free medium containing GPLD1-Gamillus, a lysosome-resistant GFP fusion, along with substrates. Cells were immunostained for LAMP1 (lysosomal marker) and imaged by confocal microscopy. Scale bar, 10 µm. ( C , D ) GPLD1-RFP-GFP and substrate complexes are degraded in lysosomes. HEK293 cells were treated as in ( A ) in the presence or absence of bafilomycin A 1 , then analyzed by flow cytometry (C) or immunoblotting with anti-mCherry, anti-luciferase, and anti-β-tubulin antibodies (D).

Article Snippet: Rabbit polyclonal antibodies against sfGFP, mCherry, and the ALFA tag were generated by Eurofins using full-length mCherry protein and the ALFA peptide as antigens.

Techniques: Luciferase, Cell Culture, Flow Cytometry, Fluorescence, Marker, Confocal Microscopy, Western Blot