Fig 1: Uncovering the senescence‐associated surface proteome. (a) SA‐β‐Gal activity was analyzed in WI‐38 fibroblasts at ~PDL24 (proliferating, P), after reaching replicative exhaustion at ~PDL54 (RS), or after exposure to 10 Gy of ionizing radiation and culture for an additional 10 days (IRIS). (b) Workflow for cell‐surface protein biotinylation and isolation for proteomic analysis; prepared using BioRender. (c) Overlap of the cell surface proteins elevated in RS vs P (green) and IRIS vs P (pink) after processing as in panels (a, b). (d) Top significantly (FDR <0.05) enriched GO Molecular Function terms after ShinyGO analysis performed on the shared cell surface proteins upregulated in senescent cells. (e) Heatmap illustrating the magnitude of induction in a subset of surface proteins, comparing RS to P cells and IRIS to P cells that were prepared as explained in (a). In the heatmap, 5NTD, ICAM1, DPP4, NECT2, and ITA1 are CD73, CD54, CD26, CD112, and CD49A, respectively. (f) WI‐38 cells were processed as in (a), and the levels of CD49A, CD73, CD109, CD54, CD112, CD44, HLA‐ABC, CD26, and loading control β‐Actin (ACTB) were assessed in Input (whole‐cell proteins) and Surface (cell‐surface proteins) by western blot analysis. “Input”, 10 μg of the protein lysate before pulldown; “Surface”, 5% of the eluted protein volume after pulldown. As the lysis buffer did not fully solubilize membrane proteins, only “Surface” proteins are quantified (ImageJ). Data are representative of three independent replicates.