Qproteome mammalian protein prer kit

The NPC tissues and normal tissues were washed with PBS, added with cell lysates containing with protease inhibitors, shaken at 4°C for 5 min and centrifuged at 4°C for 10 min at the rate 37100 × g. The proteins were extracted from the supernatant by using Qproteome Mammalian Protein Prer kit (QIAGEN, GmbH, Germany). Proteins (50 μg) were obtained for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then moved to nitrocellulose membranes. Following blocking with skimmed milk and incubated overnight with the following primary antibody IL-6 (Cat# ab9324, 0.4 μg/ml, Abcam, Cambridge, MA, USA), JAK (Cat# ab205223, 1: 500, Abcam, Cambridge, MA, USA), p-JAK (Cat# ab32101, 1: 1000, Abcam, Cambridge, MA, USA), STAT3 (Cat# 119352, 1:600, Abcam, Cambridge, MA, USA), p-STAT3 (Cat# ab76315, 1:2000, Abcam, Cambridge, MA, USA) and CyclinD1 (Cat# ab134175, 1: 10000, Abcam, Cambridge, MA, USA). After washing with tris-buffered saline solution containing Tween 20 (TBST) 4 times (10 min every time), the membranes were incubated with IRDye™ 700DX-labeled IgG (LI-COR Bioscience, NE, USA) antibody (dilution 1:1000, Upstate, NY, USA) at room temperature for 1 h, washed by TBST 4 times and developed with the substrates. The data were analyzed by LabWorks Image Acquisition and Analysis Software (UVP, Inc., Upland, CA, USA) to obtain the relative protein concentration.

After washing with phosphate buffered saline (PBS) and the appropriate amount of cell lysates was added, the spinal cord tissues were shaken at 4°C for 5 min and centrifuged at 4°C for 10 min (12000 × g). The supernatant was collected and protein was extracted using the Qproteome Mammalian Protein Prer kit (QIAGEN, GmbH, Germany). Once the corresponding protein concentration was detected and trimmed, burning and a total of 6 × buffer was added and stored at –20°C. The 50 μg of protein was conducted using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis and was transferred to a nitrocellulose filter (NC filter) using a siphon action. After sealing with skimmed milk, the mouse anti-human monoclonal antibodies GAP-43, SDF-1, CXCR4, NGF, BDNF and GFAP (1:200) were added to the protein and incubated overnight. TBST was used to wash the membrane 4 times for 10 min each time and the substrate was used. After adding goat anti-mouse IgG (1:10000) tagged with IRDyeTM800DX they were incubated at room temperature for 1 h. All antibodies were purchased from Upstate Biotechnology, Inc. (NY, USA). Protein bands were analyzed and processed using the LabWorks Image Acquisition and Analysis Software (UVP, Inc., Upland, CA, USA) to obtain the relevant protein concentration.