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