Anti p erk1 2 pt202 py204 20a

Whole blood was fixed with 16% formaldehyde (Thermo Scientific) for 10 min at room temperature. Pre-warmed lysis and permeabilization buffer (0.114% Triton X-100 in PBS) was then added and the samples incubated for 15 min at 37 °C. The cells were then washed in cold wash buffer (4% FBS in PBS), and re-suspended in ice-cold 50% MetOH in PBS for 10 min at 4 °C. The cells were then washed with cold wash buffer, and then stained with anti-CD14 [RMO52], (#A22331, IOTest), anti-CD16 [B73.1] (#12-0167-42, eBioscience), CD45 [HI30] (#45-0459-42, eBioscience) and anti-p-ERK1/2 (pT202/pY204) [20A] (#562644; BD Biosciences) or anti-p-p65 (p-S529) [K10-895.12.50] (#558422; BD Biosciences) in wash buffer for 30 min at room temperature.

For the ERK1/2 phosphorylation assay, lymphocytes from LN of R7-I, -II, or -III mice were isolated and stained with anti-CD8 (5H10, Invitrogen) and anti-CD4 (GK1.5, BioLegend) antibodies. Splenocytes loaded with ROP7 peptide were used as stimulators. Lymphocytes were then stimulated by addition of splenocytes and incubated for 0, 1, 2, 4, 8, and 12 min at 37°C. At the indicated time points, cells were fixed with paraformaldehyde at a final concentration of 2%. Cells were permeabilized by addition of ice-cold 90% methanol and stored over night at −20°C. Next, cells were washed and stained with anti-pERK1/2 (pT202/pY204) (20A, BD Biosciences) and acquired using an LSR II flow cytometer. Data were analyzed using FlowJo and Prism software.