Anti phospho erk 197g2

Cells were lysed with lysis buffer (50 mM Tris-HCl [pH7.5], 150 mM NaCl, 10 mM MgCl₂, 0.5% Nonidet P-40) containing protease and phosphatase inhibitor cocktail (Thermo Scientific). Antibodies used for Western blotting are as follows: anti-phospho-ERK (197G2, Cell Signaling) anti-Ras (Ras-10, Millipore) anti-Rasal3 (homemade poly-clonal Rabbit IgG). Horseradish peroxidase-conjugated anti-IgG secondary antibodies against rabbit or mouse IgG (GE) were used with Lumiglo substrate (Cell signaling).

For cell-surface staining, the following Abs were used: anti-CD25 (PC61), anti-CD62L (MEL-14), anti-CD4 (RM4-5 and GK1.5), anti-CD8α (5H10-1 and 53-6.7), anti-Foxp3 (FJK-16a), anti-TCRβ (H57-597), anti-CD69 (H1.2F3), anti-CD44 (IM7) Abs. All antibodies were purchased from eBioscience or Biolegend. To examine ERK phosphorylation, cells were fixed and permeabilized with 90% methanol and stained with anti-phospho-ERK (197G2, Cell Signaling) Ab. Foxp3 staining was performed using the FOXP3 staining kit (00-5523-00, eBioscience) according to the manufacturer’s protocol. Cells were analyzed using a FACS CantoII (Becton Dickinson), and the data were analyzed using FlowJo software (Tree Star).