Fig. 3: Programmable target stabilization via language model-derived peptides.

A Programmable target stabilization via language model-derived peptides. Created in BioRender. Chatterjee, P. (2025) https://BioRender.com/h25h541. B FOXP3-mCherry stabilization in HEK293T cells. Cellular mCherry fluorescence was measured by flow cytometry in independent replicates (n = 3). Normalized cell fluorescence was calculated by dividing the percentage of mCherry+ cells in a sample by that of control cells expressing a duAb vector expressing a non-specific poly-glycine (polyG) control peptide sequence. C Stabilization of endogenous WEE1 in protein extracts of HepG2 cells analyzed by immunoblotting. Cells were transiently transfected with a pcDNA3 plasmid encoding a polyG-OTUB1 control or one of the peptide-guided OTUB1 constructs as indicated. Transient transfection with an empty pcDNA3 plasmid served as an additional control. Blots were probed with anti-WEE1 and anti-GAPDH antibodies and are representative of biological replicates (n = 3) and technical replicates (n = 2) with similar results. D Stabilization of endogenous PAX3::FOXO1 in protein extracts of RH4 cells analyzed by immunoblotting. RH4 cells were transiently transfected with a pcDNA3 plasmid encoding one of the candidate duAbs while transfection with a polyG peptide-guided duAb served as a control. Blots were probed with anti-FOXO1 and anti-GAPDH antibodies and are representative of biological replicates (n = 3). For all immunoblots in (C) and (D), an equivalent amount of protein was loaded in each lane. Molecular weight (MW) ladder is indicated at left. Intensity of target protein bands was calculated via densitometry and normalized to intensity of GAPDH loading control and then normalized to polyG-OTUB1 control. Data are the average of biological replicates and technical replicates (n = 3 for WEE1 and PAX3::FOXO1). Statistical analysis for this figure was performed using the two-tailed Student’s t-test using GraphPad Prism 10 software, with calculated p values are represented as follows: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. The p values above each bar in the fold stabilization and densitometry analyses represent the comparison between the control (polyG-OTUB1, no DUB inhibitor) and the respective sample; all other p value notations compare the specified samples. Please refer to source data for numeric p values. All structures were predicted via the AlphaFold3 server, and the shading was done according to AlphaFold’s confidence metric, plDDT, as follows: Very low (plDDT <50) = Orange, Low (70 > plDDT > 50) = Yellow, Confident (70 > plDDT > 90) = Light Blue, Very high (plDDT > 90) = Light Blue.