Western blot analysis of phosphorylated ERK1/2 expression and PAR formation

Freshly isolated lymphocytes were incubated with H₂O₂, PMA (50 ng/ml) or PBS at 37°C. In some experiments, lymphocytes were pretreated with a MEK inhibitor (25 µM PD98059), a PARP inhibitor (2 µM PJ34), DMSO (0.05%) or medium before exposure to H₂O₂ or PBS. To prepare lysates, stimulated cells were disrupted in RIPA buffer (Sigma-Aldrich) containing a protease inhibitor cocktail (Sigma Aldrich). The cell lysate was clarified by centrifugation at 8,000×g for 10 min at 4°C to pellet cell debris. The protein concentration of the cell lysate was determined using the Pierce BCA protein assay kit (Pierce Biotechnology, Rockford). The lysate was then mixed with NuPAGE LDS sample buffer (Invitrogen) and NuPAGE sample reducing agent (Invitrogen), heated at 70°C for 10 min, and resolved on 4–12% NuPAGE Novex Bis-Tris precast gels (Invitrogen). Electrophoresis was conducted at 200 V for 50 min and the proteins were transferred to nitrocellulose membranes using iBlot Gel Transfer Device (Invitrogen). The membranes were blocked with buffered saline containing detergent and Hammersten casein solution (Invitrogen), and then washed with PBS-Tween 20. For detection of phosphorylated ERK (p-ERK), membranes were incubated with rabbit anti phospho-ERK1/2 Ab (Cell Signaling Technology, MA, USA) or rabbit total ERK1/2 (Cell signaling technology) followed by incubation with a HRP-conjugated goat anti-rabbit Ab (DAKO). For PAR detection, membranes were incubated with mouse anti-poly ADP ribose mAb (BD) or mouse anti-β tubulin (Invitrogen) followed by incubation with a HRP-conjugated polyclonal rabbit anti-mouse Ab (Dako, Denmark). The signals were visualized using enhanced chemiluminescence (Chemi-Doc, Bio-Rad) with ImmunoStar HRP detection kit (Bio-Rad, Hercules, CA). The intensities of the bands were quantified using the NIH ImageJ [47] software.