Cell surface–bound IFNγ mediates persistent signaling regardless of cytokine manufacturer and can be degraded by trypsinization. (A) Quantification of pSTAT1 intensity normalized to 3H IFNγ in Fig. 2 G. (B) Human macrophages were treated with Escherichia coli sourced IFNγ purchased from PeproTech and mammalian-sourced IFNγ purchased from Sigma-Aldrich and ACRO. Cells were treated at 100 ng/ml for 8 h, washed, and cultured for an additional 48 h in regular media prior to collection. Cells were collected for immunoblot at the indicated times. Representative blot of duplicates from one human subject. (C) Quantification of pSTAT1 band intensity normalized to tubulin at each time point in B. (D) Human A549 airway epithelial cells were stimulated with 100 ng/ml IFNγ, washed, and cultured for an additional 72 h. Cells were collected for immunoblot at the indicated timepoints. Representative blot of two replicates. (E) Quantification of pSTAT1 band intensity normalized to tubulin at each time point in D. (F) Human macrophages were treated with 100 ng/ml IFNγ for 8 h, washed, and lifted by scraping after incubation with either PBS, 0.5 mM EDTA in PBS, or trypsin. Cells were replated and cultured for an additional 24 h in regular media. Cells were collected for immunoblot at the indicated times. (G) Quantification of pSTAT1 band intensity normalized to GAPDH for each condition in F. Statistical tests were determined by single-tailed t test. *P < 0.05; **P < 0.01. Source data are available for this figure: SourceData FS3.