Journal: bioRxiv

Article Title: Targeted protein degradation in lysosome utilizing naturally produced bifunctional antibodies with high levels of mannose 6-phosphate glycans

doi: 10.1101/2024.09.03.611037

Figure Lengend Snippet: A . Schematic showing the procedure for detection of FLAG-mCherry and NCA-FLAG or PNCA-FLAG complexes binding toward CI-MPR. B . Bound mCherry fluorescence from (A) for NCA-FLAG (gray) or PNCA-FLAG (red). N=3 or 4. C . HepG2 cells were incubated with FLAG-mCherry and NCA-FLAG or PNCA-FLAG overnight with or without 2 mM M6P and cells were examined by fluorescent microscopy to detect mCherry signal (red). Lysosomes were stained with LysoTracker (green) and Hoechst (blue) for nuclei. D-H . Cell lines (D. HEK293T, E. Tay-Sachs Patient Fibroblasts, F. Daoy, G. HepG2, or H. MDA-MB-231) were incubated with FLAG-HexM enzyme with NCA-FLAG or PNCA-FLAG or with addition of 2 mM M6P. HexM internalization was determined by performing a HexM activity assay and activity graphed – HexM alone (gray), NCA-FLAG with HexM (Green), PNCA-FLAG with HexM (Blue), M6P added (black striped bars). Background HexM activity in untreated group wa subtracted. Dot represents each independent experiment. N=3-6. I . HEK293T CI-MPR -/- cells were generated by CRISPR-Cas9. Western blot shows CI-MPR deficiency in the cells. J . Graphs depict internalized HexM activity in CI-MPR -/- cells. Dot represents each independent experiment. N=3. Data are represented as mean values; error bars represent the standard deviation of biological replicates.

Article Snippet: HepG2 (ATCC, HB-8065), Daoy (ATCC, HTB-186) and A431 (Sigma, 85090402) cells were cultured in MEM medium (Corning, 10-009-CV) with 10% FBS.

Techniques: Binding Assay, Fluorescence, Incubation, Microscopy, Staining, Activity Assay, Generated, CRISPR, Western Blot, Standard Deviation

Journal: bioRxiv

Article Title: Targeted protein degradation in lysosome utilizing naturally produced bifunctional antibodies with high levels of mannose 6-phosphate glycans

doi: 10.1101/2024.09.03.611037

Figure Lengend Snippet: A . Schematic demonstrating production of Ab-TNFα and PNCA-TNFα in CHO cells. B . Western blotting to examine the Ab-TNFα and PNCA-TNFα heavy chain N-glycosylation after Endo-H and PNGase-F treatment. C-D . CI-MPR affinity chromatograph to determine the binding of Ab-TNFα and PNCA-TNFα. To visualize the binding signal, Alexa Fluor 594 conjugated to Ab-TNFα (C) or PNCA-TNFα (D) was used for the assay. Red dash line indicates the concentration of free M6P for antibody elution from the CI-MPR column. E . Schematic depicting internalization and degradation of extracellular TNFα b PNCA-TNFα through CI-MPR to the lysosome. F . HepG2 cells were incubated with mCherry-TNFα and Ab-TNFα or PNCA-TNFα overnight with or without 2 mM M6P and cells were examined by fluorescent microscopy to detect mCherry-TNFα signal (red). Lysosomes were stained with LysoTracker (green) and Hoechst for Nuclei (blue). G . Internalized mCherry-TNFα protein was analyzed by western blot for cell lysates using antibodies to detect mCherry, TNFα, and GAPDH as a loading control. H . Western blot analysis for internalized mCherry-TNFα with the addition of 1% or 0.5% of lysosomal protease inhibitors (PI). Antibodies are used to detect mCherry, TNFα and GAPDH, as a loading control.

Article Snippet: HepG2 (ATCC, HB-8065), Daoy (ATCC, HTB-186) and A431 (Sigma, 85090402) cells were cultured in MEM medium (Corning, 10-009-CV) with 10% FBS.

Techniques: Western Blot, Glycoproteomics, Binding Assay, Concentration Assay, Incubation, Microscopy, Staining, Control