protein concentration  (R&D Systems)

Journal: bioRxiv

Article Title: Sheddase Targeting Chimeras (SHEDTACs) catalyze membrane target proteolysis

doi: 10.64898/2026.02.06.703938

Figure Lengend Snippet: ( a ) Design of a SHEDTAC library spanning five anti-ADAM10 VHH and three anti-LAG3 VHH, all targeting distinct epitopes. Diverse configurations are achieved through N→C or C→N terminal VHH fusions, affording a library of thirty unique LAG3/ADAM10 bispecific combinations. ( b ) Reducing SDS-PAGE analysis of purified SHEDTACs used to treat cells in ( c,d ). ( c ) T cell surface LAG3 shedding by the protease ADAM10, which is accelerated by SHEDTACs ( d ) Western blot analysis of T cell pellets indicating levels of intact LAG3 on cells following 24h treatment, quantified in ( e ). Intensity is expressed as percent of vehicle (V). “+” indicates the addition of ionomycin (10µg/ml) to induce ADAM10 activity.

Article Snippet: The following day, the membrane was incubated for 1h with a 5ml volume of polyclonal goat IgG anti-human LAG3 (R&D Systems, AF2319) and rat anti-human GAPDH (Biolegend, 607902) each diluted 1:1000 in PBS containing 0.1% w/v BSA in PBST.

Techniques: SDS Page, Purification, Western Blot, Activity Assay

Journal: bioRxiv

Article Title: Sheddase Targeting Chimeras (SHEDTACs) catalyze membrane target proteolysis

doi: 10.64898/2026.02.06.703938

Figure Lengend Snippet: ( a ) Reducing SDS-PAGE indicating SHEDTAC#8, selected for its high activity shown in . ( b-e ) Flow cytometry contour plots showing LAG3 abundance on CD3+ADAM10+ PBMCs following 1h treatment with ( b ) vehicle, ( c ) 500nM SHEDTAC, ( d,e ) equimolar TEV-proteolyzed SHEDTAC, serving as monospecific controls. ( f ) Flow cytometry contour plots showing LAG3 abundance on CD3+ADAM10+ PBMCs treated with SHEDTAC (left) or equimolar TEV-digested SHEDTAC (right). Prior to treatment, cells were incubated for 2h at 37°C with vehicle (left plots), proteasome inhibitor (MG132, 10µM, middle plots), or lysosome inhibitor (Dynasore, 50µM, right plots). ( g ) Western blot analysis of cell pellets and conditioned cell supernatants treated with SHEDTAC, sampled every 10 minutes for 60 minutes, indicating time-dependent decreases in full-length (∼70kDa) LAG3 and concomitant increases in soluble LAG3 (sLAG3, ∼60kDa) ectodomain released into the growth medium by ADAM10. ( h ) quantification of data from ( g ) normalized to GAPDH and expressed as percent control of cell pellet at t=0. ( i ) Cells from ( g ) following 24h SHEDTAC treatment. ( j ) Ratio of LAG3:ADAM10 as determined by flow cytometry over a range of SHEDTAC concentrations.

Article Snippet: The following day, the membrane was incubated for 1h with a 5ml volume of polyclonal goat IgG anti-human LAG3 (R&D Systems, AF2319) and rat anti-human GAPDH (Biolegend, 607902) each diluted 1:1000 in PBS containing 0.1% w/v BSA in PBST.

Techniques: SDS Page, Activity Assay, Flow Cytometry, Incubation, Western Blot, Control

Journal: bioRxiv

Article Title: Sheddase Targeting Chimeras (SHEDTACs) catalyze membrane target proteolysis

doi: 10.64898/2026.02.06.703938

Figure Lengend Snippet: ( a ) LAG3 suppresses T cell signaling through homodimer formation, and interactions with the TCR on T cells and MHCII on antigen presenting cells (APCs) (left). LAG3-SHEDTACs catalyze LAG3 proteolysis by endogenous protease ADAM10 to restore TCR signaling and induce a luciferase reporter (right). ( b ) Flow cytometry contour plots indicating LAG3 abundance on ADAM10(+) luciferase reporter Jurkat cells treated with isotype control or LAG3-SHEDTAC. ( c ) Dose-dependent luminescence increases following treatment with SHEDTAC at the indicated concentration, illustrating enhanced TCR signaling that is afforded through LAG3 shedding. RLU = relative luminescence units

Article Snippet: The following day, the membrane was incubated for 1h with a 5ml volume of polyclonal goat IgG anti-human LAG3 (R&D Systems, AF2319) and rat anti-human GAPDH (Biolegend, 607902) each diluted 1:1000 in PBS containing 0.1% w/v BSA in PBST.

Techniques: Luciferase, Flow Cytometry, Control, Concentration Assay

Journal: bioRxiv

Article Title: Sheddase Targeting Chimeras (SHEDTACs) catalyze membrane target proteolysis

doi: 10.64898/2026.02.06.703938

Figure Lengend Snippet: Gating strategy for activated CD3+ADAM10+LAG3+ PBMCs

Article Snippet: The following day, the membrane was incubated for 1h with a 5ml volume of polyclonal goat IgG anti-human LAG3 (R&D Systems, AF2319) and rat anti-human GAPDH (Biolegend, 607902) each diluted 1:1000 in PBS containing 0.1% w/v BSA in PBST.

Techniques:

Journal: bioRxiv

Article Title: Sheddase Targeting Chimeras (SHEDTACs) catalyze membrane target proteolysis

doi: 10.64898/2026.02.06.703938

Figure Lengend Snippet: Flow cytometry TEV-normalization scheme. SHEDTACs were normalized to their equimolar TEV-proteolyzed controls, and significant shedding was indicated wherever this ratio ‘x’ was x<1. In contrast, TEV-normalized shedding where x≥1 indicates low SHEDTAC activity, or VHH competition with LAG3 detection reagents, confirmed by western blot

Article Snippet: The following day, the membrane was incubated for 1h with a 5ml volume of polyclonal goat IgG anti-human LAG3 (R&D Systems, AF2319) and rat anti-human GAPDH (Biolegend, 607902) each diluted 1:1000 in PBS containing 0.1% w/v BSA in PBST.

Techniques: Flow Cytometry, Activity Assay, Western Blot

Journal: bioRxiv

Article Title: Sheddase Targeting Chimeras (SHEDTACs) catalyze membrane target proteolysis

doi: 10.64898/2026.02.06.703938

Figure Lengend Snippet: Comparison of western blot versus flow cytometry analyses to assess LAG3 shedding by ADAM10 following treatment with SHEDTACs

Article Snippet: The following day, the membrane was incubated for 1h with a 5ml volume of polyclonal goat IgG anti-human LAG3 (R&D Systems, AF2319) and rat anti-human GAPDH (Biolegend, 607902) each diluted 1:1000 in PBS containing 0.1% w/v BSA in PBST.

Techniques: Comparison, Western Blot, Flow Cytometry

Journal: bioRxiv

Article Title: Sheddase Targeting Chimeras (SHEDTACs) catalyze membrane target proteolysis

doi: 10.64898/2026.02.06.703938

Figure Lengend Snippet: ( a ) Soluble LAG3 (sLAG3) generation through receptor shedding. ( b ) primary amino acid sequence analysis showing transmembrane and intracellular regions totaling ∼8.8kDa. ( c ) Dose-dependent loss of LAG3 abundance on T cells following treatment with SHEDTACs. Contour plots correspond to data plotted in . ( d ) Concomitant soluble LAG3 production in conditioned supernatants from cells treated in ( c ).

Article Snippet: The following day, the membrane was incubated for 1h with a 5ml volume of polyclonal goat IgG anti-human LAG3 (R&D Systems, AF2319) and rat anti-human GAPDH (Biolegend, 607902) each diluted 1:1000 in PBS containing 0.1% w/v BSA in PBST.

Techniques: Sequencing