PP4/PP2A activity

Protein phosphatase 4c and 2A activities were measured by measuring phosphatase activity (PP2A phosphatase activity kit, Millipore, CA, USA) on DRGs after central and peripheral injury prior to PP4 or PP2A immunoprecipitation. Briefly, sciatic DRGs were extracted 24 h after surgery, protein lysates were obtained with 1 litre of imidazole buffer (20 mM imidazole‐HCl, 2 mM EDTA, 2 mM EGTA, pH 7.0 with 10 μg/ml each of aprotinin leupeptin, pepstatin, 1 mM benzamidine and 1 mM PMSF) per 25 g of tissue, homogenized with micropestles on ice and centrifuged at 2,000 g for 5 minutes at 4°C. Protein lysate concentration was measured by BCA, and 100 μg of protein was used for each reaction, together with 4 μg of PP2A (Millipore, CA, USA), PP4 (PPX, Santa Cruz, CA, USA) antibodies or Mouse IgG (Millipore, CA, USA). Samples were processed following the kit instructions, and activity of samples was then measured in an Infinite M200 Pro Microplate Reader (Tecan) at 650 nm according to the manufacturer's guidelines.