Phosphorylation Analysis of Immune Cells

Phosphorylation status of p65 and c-JUN were analyzed by flow cytometry in isolated CD4 T cells (StemCell Technologies) as previously described (33 (link)). Briefly, cells were stimulated for 15 minutes at 37°C in the absence or presence of 2.5µM ALN, 2.5µg/mL PAM, 1µg/mL ZOL or 50nM PMA as a positive control. Cells were washed and stained with a viability dye (Zombie Aqua Fixable viability kit, BioLegend) and fixed with 100µl of pre-warmed Cytofix Fixation Buffer (BD Biosciences) for 10 minutes at 37°C. Cells were permeabilized while vortexing with 100 µL of prechilled Perm Buffer III (BD Biosciences) and incubated for 30 minutes on ice. After incubation, cells were washed and stained with anti-NFkB p65 (pS529, clone K10-895.12.50, BD Biosciences) or Phospho-c-Jun (Ser73, clone D47G9, Cell Signaling Technology) for 16 hours at 4°C. After washing, samples were acquired on a BD LSR Fortessa TM X-20 instrument (BD Biosciences) and analyzed using Flowjo (FlowJo v.10.8.1).