Fig. 5

Expression of total and phosphorylated Lck and ZAP70 proteins in CCR4⁺CCR6⁺ Th17 versus CXCR3⁺CCR6⁻ Th1 cells. Matched Th17 and Th1 subsets were isolated and stimulated by CD3/CD28 Abs for 3 days as described in Fig. 1. Cells were fixed on poly-l-lysine coated slides. Intracellular staining was performed with rabbit anti-human Abs against total or phosphorylated Lck (a, b) and ZAP70 (c, d). DAPI was used to stain cell nuclei. Slides were observed by fluorescence microscopy using a spinning-disc zeiss cell observer microscope. The visualization of total Lck (a) and ZAP70 (c) expression was performed using a 100× oil objective (NA = 1.46) in a spinning-disc confocal mode. Shown are maximum intensity z projection of z stack of matched Th17 and Th1 cells from one donor representative of observations made with cells from two different donors. The relative expression of total and phosphorylated Lck (b) and ZAP70 (d) was further quantified using the Image J software after observations made by epifluorescence with a 40× oil objective (NA = 1.40). b, d Shown are expression of total-Lck and total-ZAP70 (upper panels) together with phosphosphorylated Lck (Tyr 394, Tyr 505) and ZAP70 (middle and lower panels) in matched Th17 versus Th1 cells from two different donors (n = 50–100 cells per subsets). Horizontal red lines indicate median values. Unpaired t test p values are indicated on the figures