Extended Data Fig. 5: Validation of two model targets of proteasomally-cleaved peptides by the dTAG system.
From: Cell-autonomous innate immunity by proteasome-derived defence peptides
a, Bar graph showing S. enterica growth in conditioned medium from A549 cells expressing shCTRL or shPPP1CB at the final time point (4.5 h). mean ± s.e.m. (n = 6 biological replicates). Unpaired two-tailed t-test, ns P = 0.8778. b, Western blot analysis of PPP1CB levels in A549 cells expressing shCTRL or shPPP1CB, normalized to Actin. c, Western blot of PPP1CB degradation using the dTAG system across time points (1–6 h). d, Sequence alignments of PPP1CB peptides identified by MAPP. Below (scores <2), above (scores > 2). Zoomed-in view highlights peptides with scores > 5. e, Bar graphs showing S. enterica growth in conditioned medium from A549 dTAG cells at the final time point (4.5 h) for GFP (left) and PPP1CB (right). mean ± s.e.m. (n = 4 biological replicates, 2 or 3 technical replicates). Unpaired two-tailed t-test, ns P = 0.6338, ** P = 0.0073. f, Schematic representation of the dTAG peptidomics experiment. g, Spectra and MS/MS ions by which the PPP1CB-derived peptide was identified in the A549 secretome following dTAG-PPP1CB degradtation. h, Western blot analysis of HA-PSMG2 levels after 6 h with DMSO or dTAGV−1. i, Bar graph of S. enterica growth in conditioned medium from A549 cells expressing dTAG-GFP or dTAG-PSMG2 treated with DMSO or dTAGV−1. mean ± s.e.m. (n = 6; 2 biological replicates). Unpaired two-tailed t-test, ** P = 0.0068. j-l Imaging of A549 cells expressing dTAG-PSMG2 treated with DMSO or dTAGV-1, infected with RFP- S. typhimurium (magenta), and stained with DAPI (blue). j, Representative images k, Bar plot showing quantification of RFP- S. typhimurium per nucleus. mean ± s.e.m. (n = 4 biological replicates, five images per replicate). Unpaired two-tailed t-test, *** P = 0.0002. l, Bar plots showing quantification of RFP/Salmonella per nucleus in A549 cells dTAG PSMG2. Five images per biological replicate ± s.e.m. (n = 4 independent samples) Unpaired two-tailed t-test, *** P = 0.0002. Illustrations created in BioRender. Merbl, Y. (2025) https://BioRender.com/x10a923.
