Flow cytometric analysis of pSTAT1 [14 (link)] and liquid chromatography-tandem mass spectrometry (LC-MS-MS) analysis [16 (link)] of nitrated STAT1 (nSTAT1) were conducted on all samples. For flow cytometry studies, frozen PBMCs were thawed at 37 °C, washed with culturing media, and allowed to rest overnight in complete media at 5% CO2 at 37 °C. PBMCs were stimulated with interferon-α (Miltenyi Biotec, Cambridge, MA, USA) at 0, 102 U/mL, and 104 U/mL and incubated for 15 min in media, as previously described [10 (link)].
The live/dead marker Zombie NIR (BioLegend, San Diego, CA, USA) was used before permeabilization to distinguish live cells. The samples were permeabilized using the FIX & PERM Cell Permeabilization Kit with methanol modification (Fisher Scientific, Hampton, MA) and fixed at −20 °C for a minimum of 2 h. pSTAT1 was detected by a pSTAT1 antibody (AF488; BD Biosciences, San Jose, CA, USA). Flow cytometry data were collected either on Canto or LSRII flow cytometers (BD Biosciences, San Jose, CA, USA), and data were analyzed in FCS Express (De Novo Software, Pasadena, CA, USA). Measurement of patient-derived PBMCs for nSTAT1 and native STAT1 via LC-MS-MS SRM experiments was performed as described previously [16 (link)].
The live/dead marker Zombie NIR (BioLegend, San Diego, CA, USA) was used before permeabilization to distinguish live cells. The samples were permeabilized using the FIX & PERM Cell Permeabilization Kit with methanol modification (Fisher Scientific, Hampton, MA) and fixed at −20 °C for a minimum of 2 h. pSTAT1 was detected by a pSTAT1 antibody (AF488; BD Biosciences, San Jose, CA, USA). Flow cytometry data were collected either on Canto or LSRII flow cytometers (BD Biosciences, San Jose, CA, USA), and data were analyzed in FCS Express (De Novo Software, Pasadena, CA, USA). Measurement of patient-derived PBMCs for nSTAT1 and native STAT1 via LC-MS-MS SRM experiments was performed as described previously [16 (link)].
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