Protein phosphatase inhibitor cocktail

The cellular proteins from cultured cells were extracted by lysing in lysis buffer (50 mM Tris-HCl pH 7.5, 125 mM NaCl, 5 mM EDTA and 0.1% Triton X-100) containing 1% protease inhibitor and 1% protein phosphatase inhibitor cocktail (Sigma-Aldrich). Western blot was performed following previously reported methods [11 (link)]. Briefly, proteins were electrophoresed in a 5–20% sodium dodecyl sulfate-polyacrylamide gradient gel. After electrophoresis, proteins were transferred to the PVDF membrane with an iBlot^® Gel Transfer Stack (Invitrogen). The membrane was blocked using 5% skim milk and then incubated overnight at 4 °C with a primary antibody. After washing, the membrane was incubated with a secondary antibody for 90 min at 25 °C. Chemiluminescence was excited by ImmunoStar^® Reagents (FUJIFILM Wako Pure Chemical) and captured by the ImageQuant^TM LAS 4000 mini (FUJIFILM).
The primary antibodies used were phosphorylated (p)-Stat3 Tyr705 (#9145, Cell Signaling Technology; CST, Beverly, MA, USA), total (t)-Stat3 (#4904, CST), pAkt Ser473 (#4060, CST), tAkt (#9272, CST), pp38 MAPK (#4511, CST), tp38 MAPK (#8690, CST), CXCR1 (#MAB330, R&D Systems), CXCR2 (#ab65968, Abcam, Cambridge, UK), and β-actin (#4970, CST). The signal intensities of bands were quantified using the ImageJ 1.53t.

Trichostatin A and okadaic acid were obtained from Wako Pure Chemical Industries. The protease inhibitor cocktail was from Nacalai Tesque and the protein phosphatase inhibitor cocktail was from Sigma. Nicotinamide was from Kanto Chemical.

“Nerve bridge” tissues (from four sciatic nerves) or cells were homogenized in radio immunoprecipitation assay buffer (RIPA) 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS, 50 mM Tri-HCl, pH 8.0) with Protease Inhibitor Cocktails (Sigma-Aldrich), phenylmethylsulfonyl fluoride (Amresco, Solon, Cleveland, OH), and protein phosphatase inhibitor cocktail (Sigma-Aldrich). Tissue lysate was processed using standard SDS-polyacrylamide gel electrophoresis and western blot. Highly sensitive chemiluminescence (ECL kit from Millipore) was used to illustrate the bands. Images were obtained through X-ray film exposure system. Densitometric analysis of the bands was performed using NIH Image J software package (http://rsb.info.nih.gov/ij/). Primary antibodies used were as follows: rabbit anti-AKT1 monoclonal antibody (1 : 3000; Cell Signaling), rabbit anti-phosphorylated AKT monoclonal antibody (S473, 1 : 2000; Cell Signaling), rabbit anti-phosphorylated S6 (1 : 2000, Cell Signaling), rabbit anti-phosphorylated mTOR (1 : 1000; Cell Signaling), and rabbit anti-MBP polyclonal antibody (1 : 1000; Abcam). The horseradish peroxidase conjugated secondary antibodies were all purchased from Millipore and used at 1 : 10,000–1 : 50,000 dilution.